-
PYRIDOXAL PHOSPHATE ENZYMES:Mechanistic, Structural, and
EvolutionaryConsiderations
Andrew C. Eliot1 and Jack F. Kirsch21Department of Chemistry and
2Departments of Chemistry and Molecular and CellBiology, University
of California, Berkeley, California 94720-3206;
email:[email protected], [email protected]
Key Words substrate specificity, reaction type specificity,
enzyme inhibition,enzyme mechanism
f Abstract Pyridoxal phosphate (PLP)-dependent enzymes are
unrivaled in thediversity of reactions that they catalyze. New
structural data have paved the way fortargeted mutagenesis and
mechanistic studies and have provided a framework forinterpretation
of those results. Together, these complementary approaches yield
newinsight into function, particularly in understanding the origins
of substrate andreaction type specificity. The combination of new
sequences and structures enablesbetter reconstruction of their
evolutionary heritage and illuminates unrecognizedsimilarities
within this diverse group of enzymes. The important metabolic roles
ofmany PLP-dependent enzymes drive efforts to design specific
inhibitors, which arenow guided by the availability of
comprehensive structural and functional databases.Better
understanding of the function of this important group of enzymes is
crucial notonly for inhibitor design, but also for the design of
improved protein-based catalysts.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . 384MECHANISTIC VERSATILITY OF PLP . . .
. . . . . . . . . . . . . . . . . . . . . . 385STRUCTURAL DIVERSITY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
387DETERMINANTS OF REACTION TYPE . . . . . . . . . . . . . . . . .
. . . . . . . . 394
Dunathan Stereoelectronic Hypothesis . . . . . . . . . . . . . .
. . . . . . . . . . . . 394Transamination . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
395Racemization . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 398Role of the Closed Conformation .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
DETERMINANTS OF SUBSTRATE SPECIFICITY. . . . . . . . . . . . . .
. . . . . 399Role of the Closed Conformation . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . 399Dual Specificity of
Aminotransferases . . . . . . . . . . . . . . . . . . . . . . . . .
. 399
EVOLUTION . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 403MECHANISMS OF INHIBITION . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 406
Annu. Rev. Biochem. 2004. 73:383–415doi:
10.1146/annurev.biochem.73.011303.074021
Copyright © 2004 by Annual Reviews. All rights reservedFirst
published online as a Review in Advance on April 2, 2004
3830066-4154/04/0707-0383$14.00
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Noncovalent Inactivation . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . 406Activated Nucleophiles . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
408Activated Electrophiles . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 409Reversible Competitive Inhibition
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410The
Challenge of Inhibitor Specificity . . . . . . . . . . . . . . . .
. . . . . . . . . . 411
CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . 411
INTRODUCTION
Following its identification in 1951 as one of the active
vitamers of vitamin B6(1), pyridoxal 5�-phosphate (PLP) has been
the subject of extensive researchdirected toward understanding its
unequaled catalytic versatility.1 As a result, thebasic mechanisms
of PLP-assisted reactions, both in solution and enzyme-associated,
have been well characterized and are now a staple of
biochemistrytextbooks (for example, see 2, 3). A number of reviews
have addressed thesubject in greater detail, and readers are
directed particularly to the recent articlesby Hayashi (4) and John
(5). Transaminases, edited by Christen & Metzler (6),remains an
excellent and thorough source of information about these
enzymes.More detailed reviews concentrate on the function of a
number of specificenzymes, including tryptophan synthase (7),
O-acetylserine sulfhydrylase (8),�-aminolevulinate synthase (9),
serine hydroxymethyltransferase (SHMT) (10),and branched chain
amino acid aminotransferase (BCAT) (11).
In addition to their versatility as catalysts, PLP-dependent
enzymes haveattracted attention because of their widespread
involvement in cellular processes.These enzymes are principally
involved in the biosynthesis of amino acids andamino acid-derived
metabolites, but they are also found in the biosyntheticpathways of
amino sugars (12) and other amine-containing compounds.
Theirimportance is further underscored by the number identified as
drug targets. Forexample, inhibitors of �-aminobutyric acid
aminotransferase (GABA ATase) areused in the treatment of epilepsy
(13), SHMT has been identified as a target forcancer therapy (14),
and inhibitors of ornithine decarboxylase (ODC) areemployed in the
treatment of African sleeping sickness (15). Functional defectsin
PLP enzymes have furthermore been implicated in a number of
diseasepathologies, including homocystinuria, which is most
frequently caused bymutations in cystathionine �-synthase (16,
17).
1Abbreviations: AATase, aspartate aminotransferase; ACC,
1-aminocyclopropane-1-carboxylate; AlaP, 1-aminoethylphosphonate;
ALR, alanine racemase; AONS, 8-amino-7-oxononanoate synthase;
ATase, aminotransferase; BCAT, branched chain amino
acidaminotransferase; DAAT, D-amino acid aminotransferase; DGD,
dialkylglycine decar-boxylase; GABA, �-aminobutyric acid; OAT,
ornithine aminotransferase; ODC, ornithinedecarboxylase; PLP,
pyridoxal 5�-phosphate; PMP, pyridoxamine 5�-phosphate;
SAM,S-adenosyl-L-methionine; SHMT, serine hydroxymethyl transfease;
and TATase, aro-matic amino acid aminotransferase.
384 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Despite the long history of research in the field, we are only
now beginningto answer some of the most exciting questions. In
particular, technologicaladvances that have allowed ever more rapid
determination of enzyme structuresand the accumulation of large
sequence databases have also enhanced ourunderstanding of enzymatic
catalysis. Analyses of sequences and structures yieldsignificant
insight regarding the evolution of this diverse class of
enzymes.Additionally, crystal structures elegantly demonstrate how
PLP enzymes harnessthe potential of the cofactor to accelerate the
rates only of specific reactions, andthey provide the basis for
targeted mutagenesis.
This review describes many of the new insights that have come
from recentstructural and mechanistic studies, with a primary focus
on determinants ofsubstrate specificity and reaction type, as well
as the design of inhibitors for thisclass of enzyme. The mechanisms
and structures are briefly discussed to providebackground.
MECHANISTIC VERSATILITY OF PLP
PLP-catalyzed reaction types can be divided according to the
position at whichthe net reaction occurs. Reactions at the �
position include transamination,decarboxylation, racemization, and
elimination and replacement of an electro-philic R group. Those at
the � or � position include elimination or replacement.Examples of
each of these reactions are shown in Scheme 1, and the
basicmechanisms are shown in Schemes 2 (�) and 3 (� and �).
Exceptions to thesecommon types include the formation of a
cyclopropane ring from S-adenosyl-L-methionine (SAM), catalyzed by
1-aminocyclopropane-1-carboxylate (ACC)synthase (18), and the
cleavage of ACC to �-ketobutyrate and ammonia,catalyzed by ACC
deaminase (19); these are not discussed here. Because manyof the
reaction pathways share common intermediates, a number of enzymes
alsocatalyze reactions that are combinations of the basic types,
such as the decar-boxylation-dependent transamination
(aminoisobutyrate � pyruvate 3 acetone� CO2 � L-Ala) catalyzed by
dialkylglycine decarboxylase (DGD; 20) or the�-elimination and
�-replacement (O-phospho-L-homoserine � H2O 3 L-Thr �Pi) catalyzed
by threonine synthase (21).
Although the scope of PLP-catalyzed reactions initially appears
to be bewil-deringly diverse, there is a simple unifying principle.
The cofactor in all casesfunctions to stabilize negative charge
development at C� in the transition statethat is formed after
condensation of the amino acid substrate with PLP to forma Schiff
base (referred to as the external aldimine).2 The fully formed
carbanion
2There are two exceptions to this basic principle. The glycogen
phosphorylase family ofenzymes utilizes the phosphate group of PLP
for catalysis [reviewed in (106)], and theaminomutase family
catalyzes a radical-initiated reaction on PLP-bound amino
acidsubstrates [reviewed in (107)]. Neither of these families will
be discussed in this review.
385PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Scheme 1 Examples of each of the common reaction types catalyzed
by PLP-dependentenzymes. Net changes at the � carbon (top) include
racemization, decarboxylation,�-elimination and replacement, and
transamination. Net changes at the � carbon (middle)include
�-elimination or replacement, and those at the �-carbon (bottom)
are �-replacementor elimination. The basic mechanisms for these
reactions are shown in Schemes 2 and 3.
386 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
is referred to as the quinonoid intermediate (Scheme 4). The pKa
for loss of theC�-proton from an amino acid in the absence of PLP
is � 30 (22); therefore, thatanion, formation of which is required
in order to enable all of the describedchemistry, is ordinarily
inaccessible under physiological conditions.3 The stabi-lization of
the C�-anion is facilitated by delocalization of the negative
chargethrough the pi system of the cofactor, and for this reason
PLP is often describedas an electron sink. This factor allows PLP
in the absence of enzyme to catalyzemany of the possible reactions
slowly (reviewed in 6). The function of the proteinapoenzyme,
therefore, is to enhance this innate catalytic potential and to
enforceselectivity of substrate binding and reaction type. In most
cases, this selectivityis exquisite—potential side reactions are
limited to barely detectable levels.
STRUCTURAL DIVERSITY
Although PLP enzymes were for a time underrepresented in protein
structuredatabases (5), the situation has been rectified in recent
years, as an abundance ofnew structures have been solved. It was
initially postulated that the structures ofPLP enzymes would
correlate with the reaction type (24), but it has since beenfound
that each of the major structural classes contains representatives
ofmultiple reaction types, the evolutionary implications of which
are discussedbelow. All PLP enzymes whose structures have been
solved to date belong to oneof five fold types, which have been
described in detail in two recent reviews (25,26), and therefore
are only briefly summarized here. Figure 1 shows
singlerepresentatives of Fold Types I-IV, whose mechanisms are
discussed in thisreview.
The majority of known structures are of Fold Type I (aspartate
aminotrans-ferase family) enzymes, a group that includes many of
the best-characterized PLPenzymes. They invariably function as
homodimers or higher-order oligomers,with two active sites per
dimer. The active sites lie on the dimer interface, andeach monomer
contributes essential residues to both active sites. In general,
thetwo active sites are independent, but asymmetry has been
observed in a fewcases. Negative cooperativity, for example, has
been reported in GABA ATase,where a dimer with two functional
active sites exhibits the same specific activityas a dimer with
only one functional active site (27). The two active sites in
eachdimer of glutamate-1-semialdehyde aminomutase also exhibit
different reactiv-ities, as evidenced by the biphasic kinetics of
reduction of the enzyme-PLPaldimine by sodium borohydride (28).
Each monomer of Fold Type I enzymeshas a large and a small domain.
In a number of cases [e.g., aspartate amino-transferase (AATase)],
these domains move significantly upon association with
3Certain non-PLP-dependent amino acid racemases, such as
glutamate racemase (23),remain puzzling exceptions. If these
reactions proceed through a transition state that hassubstantial
anion character, it is not apparent how that state is
stabilized.
387PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
388 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
substrate, creating a closed conformation that may contribute to
the specificity forboth the substrate and the reaction type (see
below).
The structures of Fold Type II (tryptophan synthase family)
enzymes aresimilar to those of Fold Type I, but the proteins are
evolutionarily distinct (29).One significant difference is that the
active sites of Fold Type II enzymes arecomposed entirely of
residues from one monomer [first observed in tryptophansynthase
(30)]. Nevertheless, the functional form remains a homodimer
orhigher-order oligomer. These enzymes also differ from those of
Fold Type I inthat they often contain additional regulatory
domains. Examples include threo-nine synthase (31) and
cystathionine �-synthase (32), which are allostericallyregulated by
SAM, and threonine deaminase, which is regulated by isoleucineand
valine (reviewed in 33).
The Fold Type IV (D-amino acid aminotransferase family) enzymes
aresuperficially similar to Fold Types I and II, in that they are
also functionalhomodimers, and the catalytic portion of each
monomer is composed of a smalland a large domain. The cofactor is
bound in a site that is a near mirror imageof the Fold Types I and
II binding sites, so that the re rather than si face is
solventexposed (34).
Fold Types III (alanine racemase family) and V (glycogen
phosphorylasefamily) are strikingly different from the other PLP
enzymes. The Fold Type Venzymes are mechanistically distinct in
utilizing the phosphate group of thecofactor for catalysis and are
not considered further. The Fold Type III enzymesconsist of a
classical �/� barrel and a second �-strand domain. Interestingly,
themode of binding of PLP is similar to that of other fold types,
with the phosphate
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Scheme
2 Examples of mechanisms of reactions involving net change at the �
position.All reactions are shown starting with the substrate
aldimine, which is formed by transaldi-mination of the lysine-bound
PLP. Racemization: the bacterial alanine racemase utilizes
atyrosine residue (51, 104) to deprotonate L-alanine, forming the
quinonoid intermediate,which is reprotonated by a lysine residue on
the opposite face of the cofactor to produceD-alanine.
Decarboxylation: the reaction begins with loss of CO2 from the
substratealdimine, producing the quinonoid intermediate.
Protonation by an unidentified active siteresidue in ornithine
decarboxylase generates the product aldimine. �-Replacement:
thewell-studied serine hydroxymethyltransferase (10) initiates the
retro-aldol cleavage ofserine by deprotonation of the hydroxyl
group. Formaldehyde is released to generate thequinonoid
intermediate. Protonation of the quinonoid at C� by the lysine
produces theproduct aldimine of glycine. Transamination: the first
half-reaction catalyzed by tyrosineaminotransferase involves
initial proton abstraction from the glutamate aldimine at C� bythe
active site lysine, yielding the quinonoid intermediate.
Reprotonation at C4� of thecofactor by that lysine generates the
ketimine intermediate, which is subsequently hydro-lyzed to release
�-ketoglutarate, leaving the enzyme in the PMP form. A complete
catalyticcycle involves subsequent reaction with
hydroxyphenylpyruvate to give tyrosine and toregenerate the PLP
form of the enzyme.
389PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
390 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
group anchored at the N terminus of an ��helix, H-bond
interactions made to the3�-OH, and the presence of the ubiquitous
lysine Schiff base. Furthermore, theseenzymes are also obligate
dimers, as each monomer contributes residues to bothactive sites
(35).
Multiple scaffolds have clearly evolved to bind PLP and to
assist in catalysisby this cofactor. In no case does the fold type
dictate the reaction type, as eachfold type contains multiple
reaction types, and all common reaction types arefound in at least
two fold types.4
4Although only one racemase structure has been solved (alanine
racemase Fold Type III),serine racemase is predicted to be in Fold
Type II based on sequence (36), and the fungalalanine racemase in
Fold Type I (37).
Scheme 4 The primary function of PLP is to stabilize anions
generated at C�. Thenegative charge is delocalized by resonance in
the pi system of the cofactor in the quinonoidintermediate after
loss of a proton from the external aldimine.
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Scheme
3 Examples of mechanisms of reactions at the � and � positions.
�-Replacement:tryptophan synthase (7) catalyzes a net �-replacement
by first deprotonating the serinealdimine at C�, producing the
quinonoid intermediate. Protonation of the hydroxyl group bythe
active site lysine promotes its elimination, generating the
aminoacrylate aldimine.Indole adds to C� to form a second quinonoid
that is subsequently protonated at C� togenerate the product
aldimine. �-Replacement: in the cystathionine
�-synthase-catalyzed�-replacement reaction (105), O-succinyl
homoserine is deprotonated at C� to produce thequinonoid
intermediate that is subsequently protonated at C4� of the cofactor
to give theketimine intermediate. Proton abstraction at C� by an
unknown active site base results inelimination of the succinyl
group, which may occur in either a step-wise or concerted(shown)
manner. Michael addition of cysteine to the �,�-unsaturated
ketimine and subse-quent proton transfers yield a second quinonoid
intermediate that is protonated at C� to formthe product
aldimine.
391PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
392 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Fig
ure
1R
ibbo
ndi
agra
ms
ofre
pres
enta
tive
enzy
mes
ofFo
ldT
ypes
I-
IV.E
ach
stru
ctur
ede
pict
sa
hom
odim
erw
ithth
ein
divi
dual
mon
omer
sdi
stin
guis
hed
byco
lor.
The
PLP
cofa
ctor
issh
own
inre
d(t
ople
ft).
Fold
Typ
eI
(E.
coli
aspa
rtat
eam
inot
rans
fera
se;
pdb
file
1asn
)(t
opri
ght)
.Fo
ldT
ype
II(S
alm
onel
laty
phim
uriu
mO
-ace
tyls
erin
esu
lfhy
-dr
ylas
e;1o
as)
(bot
tom
left
).Fo
ldT
ype
III
(Bac
illu
sst
earo
ther
mop
hilu
sal
anin
era
cem
ase;
1sft
)(b
otto
mri
ght)
.Fo
ldT
ype
IV(t
herm
ophi
licB
acil
lus
sp.
D-a
min
oac
idam
inot
rans
fera
se;
1daa
).T
hefig
ure
was
prep
ared
with
Ras
Mol
.
393PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
DETERMINANTS OF REACTION TYPE
Dunathan Stereoelectronic Hypothesis
Well before the lack of correlation between fold type and
reaction type wasrecognized, much research was directed toward
understanding the determinantsof reaction type specificity.
Dunathan postulated in 1966 (38) that the topologyof the amino acid
aldimine determined the bond to C� that would be broken.
Hesuggested that that bond must be situated so that it will align
perpendicularly withthe pyridine ring of the cofactor in the
transition state of the reaction (Scheme 5).The ensuing carbanion
is stabilized by conjugation with the extended pi system.This
hypothesis was later confirmed when the structure of the aspartate
amino-transferase/phosphopyridoxyl aspartate complex was solved
(39). All subse-quently determined structures are consistent with
this idea. Of particular interestin this respect are enzymes that
catalyze C�-deprotonation and decarboxylationat different points
during their catalytic cycle.
8-AMINO-7-OXONONANOATE SYNTHASE (AONS) AONS, the second of
fourenzymes in the biotin biosynthetic pathway, catalyzes the
decarboxylation andaddition of a pimeloyl group to alanine (Scheme
6). Interestingly, the reactionmechanism involves initial
deprotonation rather than decarboxylation (40). Thequinonoid thus
formed attacks pimeloyl-CoA, forming a �-keto acid intermedi-ate,
which is decarboxylated and reprotonated to form the product
aldimine(Scheme 7). Decarboxylation of a �-keto acid is a facile
reaction that may notrequire the participation of the cofactor, but
the observation of the expectedquinonoid intermediate indicates
that it is likely involved (41). Although nostructure is available
of the intermediate aldimine formed prior to decarboxyl-ation, the
structure of the product aldimine shows that pimeloyl addition
occurs
Scheme 5 The Dunathan stereoelectronic hypothesis. Substrates
are bound to PLPsuch that the bond to C� that is to be broken is
aligned with the pi orbitals of thecofactor. Control of the
substrate orientation thus enables the enzyme to
distinguishbetween, for example, decarboxylation and
deprotonation.
394 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
on the face opposite from that of deprotonation, resulting in
the carboxylategroup occupying nearly the same position as that
previously held by the proton(Scheme 7).
DIALKYLGLYCINE DECARBOXYLASE (DGD) DGD is both an
aminotransferase anda decarboxylase. A complete catalytic cycle
involves decarboxylation andtransamination of dialkylglycine to
generate a ketone product and the pyridox-amine phosphate (PMP)
form of the enzyme followed by reaction with pyruvatein a typical
transamination to generate alanine and to restore the PLP form of
theenzyme (Scheme 8). Structural and mechanistic studies
demonstrated that thedecarboxylation of the dialkyl amino acids is
forced by large side chains, whichare not accommodated in the same
site as the methyl side chain of alanine. Theyinstead occupy the
same position as the alanine carboxylate. This reorientationresults
in the scissile bond being that between C� and the carboxylate
rather thanthat between C� and the proton (42) (Scheme 9). Thus
decarboxylation isprefered over deprotonation.
These examples demonstrate how PLP enzymes can strictly control
the initialbond-breakage step and limit other potential reactions
by restricting the boundsubstrate to a specific orientation.
Control of the steps subsequent to the initialbond breakage is less
well understood. C�-deprotonation, for example, can leadto �- or
�-elimination/replacement, racemization, or transamination (Schemes
2,3). To promote the desired reaction, an enzyme must rigorously
govern theelectron flow and proton transfers. The structures of PLP
enzymes have shownhow active site residues are positioned to
promote particular reaction types andto restrict possible side
reactions. These principles are readily appreciatedthrough a
comparison of the structures of enzymes catalyzing transamination
tothat of a racemase.
Transamination
The first structures of a PLP-dependent enzyme to be determined
were of themitochondrial and cytosolic aspartate aminotransferases
(AATase) (39, 43, 44).They explained much of the available
mechanistic data and inspired fruitful
Scheme 6 The reaction catalyzed by 8-amino-7-oxononanoate
synthase. The carboxylategroup of alanine is replaced by a pimeloyl
moiety. The mechanism of this reaction is shownin Scheme 7.
395PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
targeted mutagenesis investigations (45–49). The electron sink
nature of thecofactor is enhanced by a close interaction of the
pyridinium nitrogen with anaspartate residue (D222), which acts to
maintain the cofactor in the protonatedform (Scheme 10). The
displaced Schiff base lysine residue (K258) is positionedto
transfer a proton to and from C� and C4�. Since the substrates of
AATase,aspartate and glutamate, cannot undergo �- or �-
elimination, the primary sidereaction that must be minimized is
racemization. This objective is achieved byclosure of the enzyme
around the substrate, so that solvent water molecules donot access
the quinonoid intermediate to protonate the re face (50).
Furthermore,there are no active site acids in position to donate a
proton to the re face of thatintermediate. Subsequently solved
structures of other aminotransferases con-
Scheme 7 The mechanism of the 8-amino-7-oxononanoate
synthase-catalyzedreaction. The alanine aldimine is initially
deprotonated, and the resulting quinonoidattacks the pimeloyl CoA
thioester. Addition of the pimeloyl group to the faceopposite from
the deprotonation event causes the carboxylate to reorient
perpendic-ular to the pyridine ring, resulting in subsequent
decarboxylation. Protonation of thissecond quinonoid produces the
product aldimine. Adapted from Reference 41.
396 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
firmed that these are general properties of the entire class of
enzymes. TheD-amino acid aminotransferase (Fold Type IV) is
particularly interesting becauseits active site is in part a mirror
image of the L-amino acid aminotransferases (34),in that the active
site lysine is positioned on the re face and the si face is
solventexposed, neatly accounting for its opposite
stereospecificity.
Scheme 8 The reaction catalyzed by dialkylglycine decarboxylase.
In the firsthalf-reaction, a dialkyl substrate is decarboxylated
and transaminated, producingCO2, a ketone, and the PMP form of the
enzyme. The latter form subsequently reactswith pyruvate in a
transamination half-reaction to give alanine and to restore the
PLPform of the enzyme.
Scheme 9 Steric control of reaction type by dialkylglycine
decarboxylase. Thethree substituents on C� occupy distinct binding
sites, labeled A, B, and C. Whereasthe A and B sites are tolerant
of carboxylates and hydrogen or large alkyl groups,respectively,
the C site only accommodates small alkyl groups. Amino acid
substrateswith small side chains, such as alanine, bind preferably
with their carboxylatemoieties in the B site, placing the C�-proton
in the reactive A site. Substrates with alarge side chain (such as
the phenylglycine shown), however, bind with that group inthe B
site, forcing the carboxylate into the reactive A site. For this
reason, thedistinction between decarboxylation or deprotonation is
a consequence of the sub-strate structure. Adapted from Reference
42.
397PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Racemization
Although the structure of alanine racemase (ALR) from Bacillus
stearother-mophilus (51) is the only one presently available, the
catalytic mechanism isapparent from the unique architecture of its
active site. An active site Brønstedacid is expected to be on the
opposite re face to complement the lysine on the siface, and a
tyrosine provides this function here (Scheme 2). Like AATase,
ALRacts on substrates incapable of undergoing elimination;
therefore, the primaryside reaction that must be limited is
transamination. A major factor in promotingthat restriction is
likely the arginine that replaces the aspartate near the
pyri-dinium nitrogen (Scheme 10). The positively charged arginine
prevents proto-nation of the cofactor (51), thereby negating its
electron sink properties.Although it might be expected that this
interaction would also impair the normalfunction of the enzyme, the
racemization reaction may require less delocalizationof electron
density into the cofactor. In this regard, it is notable that some
knownamino acid racemases do not require a cofactor (see Footnote
1). Althoughracemization is generally presumed to proceed through a
fully formed carbanionat C� (52), the instability of this species
elicits consideration of an SE2mechanism where the incoming proton
develops some bonding with C� in aconcerted transition state.
Jencks pointed out (53) that stepwise mechanisms aregenerally
preferred because of entropy considerations, but that
mechanismsbecome concerted when the intermediate that would develop
in the stepwiseprocess is too unstable to exist.
Interestingly, some of the insights gained from this structure
cannot begenerally applied to all PLP-dependent racemases.
Paiardini et al. (54) reported
Scheme 10 Control of the electron sink properties of PLP by the
amino acid sidechain positioned nearest to the pyridinium nitrogen
atom. An aspartate residueoccupies this locus in aspartate
aminotransferase and all other known Fold Type Ienzymes, thereby
maintaining ring protonation. The corresponding residue is
anarginine in the bacterial alanine racemase, which is expected to
maintain the cofactorin the unprotonated form. Lack of a proton at
this position would greatly diminish theelectron withdrawing
properties of PLP.
398 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
an alanine racemase that is predicted to be a member of Fold
Type I and to havean aspartate residue that interacts with the
pyridinium nitrogen, as do all otherFold Type I enzymes.
Role of the Closed Conformation
Although it is not clear if the observed conformational changes
of PLP enzymescontribute to substrate specificity (see below), the
existence of the closedconformation undoubtedly contributes to
reaction type specificity. Wolfendenpointed out the advantages of a
closed conformation that makes more contactswith the bound
substrate than are possible in a conformation from which
thesubstrate must be able to dissociate (55). The closed
conformation can favorspecific reactions by providing greater
control over proton transfers and solventaccessibility. A
well-studied example is serine hydroxymethyltransferase,
whichcatalyzes a variety of side reactions (transamination,
decarboxylation, racemiza-tion, etc.) when presented with
substrates other than serine. The open form of theenzyme is unable
to discriminate between the various reaction types in the waythat
the closed form can (37, 56). Therefore, these reactions occur, at
least in part,because the substrates in question do not induce the
closed conformation.
DETERMINANTS OF SUBSTRATE SPECIFICITY
Role of the Closed Conformation
The potential contribution of induced fit to enzymatic substrate
specificity hasbeen much debated (for a recent overview, see 57),
but it is now accepted that aconformational change induced only by
certain substrates does not necessarilyresult in increased
specificity for those substrates. Thus, it has been argued thatthe
conformational change observed in AATase and many other PLP
enzymesdoes not contribute to their substrate specificities (4).
Exceptions to the generalrule, however, include cases where
substrate association or product dissociationis rate-determining
for good substrates, whereas chemistry is rate-determining forpoor
substrates (58). Since it has been shown that release of the
productoxalacetate is partially rate determining for the reaction
of AATase with aspartateand �-ketoglutarate (�KG) (59), AATase is
an example of an induced fit enzymein which the conformational
change may contribute to substrate specificity. It isnot yet clear
whether this conclusion extends to other PLP enzymes.
Dual Specificity of Aminotransferases
The basis for the dual specificity of aminotransferases has been
elucidated byrecent structural information. Because the
aminotransferase reaction requires twodifferent substrates to bind
in succession to the same cofactor in the active site,these enzymes
must be able to accommodate both structures while
discriminating
399PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
against all others. One possible solution would be for the PLP
itself to movebetween two different substrate binding sites, but
such movement has never beenobserved. An alternative is to take
advantage of flexible side chains to positionthe functional groups
into exclusive binding sites. This is the case for
histidinolphosphate aminotransferase, which reacts with both
histidinol phosphate andglutamate. The recently solved structures
of substrate complexes of this enzyme(60) show that, although the
phosphate and carboxylate groups interact primarilywith the same
arginine residue, the side chains of the two substrates have
separatebinding sites (Scheme 11).
Most known aminotransferases, however, adopt strategies for
binding bothsubstrates in the same site. Because many use the
common substrate glutamate(thereby linking other amino acids to the
cellular nitrogen pool), the problem ofdual specificity is
generally that of accommodating the negatively charged�-carboxylate
of glutamate in a site that must also accept a neutral or
positivelycharged side chain. Two common solutions to this problem
have been found: theuse of an arginine switch and an extended
hydrogen bond network.
ARGININE SWITCHES The first example of an arginine switch was
observed in anengineered enzyme that was constructed by introducing
six mutations intoAATase. These changes resulted in a substantial
increase in activity towardaromatic substrates (61). The subsequent
determination of the structure of the
Scheme 11 Dual specificity of histidinol phosphate
aminotransferase. The phosphate andcarboxylate moieties of
glutamate and histidinol phosphate, respectively, bind in the
samesite, although an additional arginine does interact with the
phosphate group. In contrast, theoppositely charged imidazole and
carboxylate side chains occupy spatially distinct sites andinteract
with different active site residues. The enzyme thus recognizes
each substratespecifically. Adapted from Reference 60.
400 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
mutant enzyme (62) showed that the large aromatic substrates are
accommodatedby movement of Arg292 out of the active site.
Dicarboxylate substrates bind their�- or �-carboxylate via direct
interaction with this arginine through a bidentatehydrogen bond/ion
pair in the canonical AATase conformation (Scheme 12). Theposition
of Arg292 is locked in AATases; therefore, substrates lacking a
carbox-ylate side chain are effectively excluded.
More recently, directed evolution techniques have been utilized
to broaden thesubstrate specificity of AATase to include branched
chain (63) or aromatic aminoacids (64). Crystal structures of
mutants with increased activity toward branchedchain amino acids
(65, 66) indicated that they also have acquired the ability to
switchArg292 out of the active site when uncharged substrates are
bound, indicating thatthis trait is easily induced in AATase and is
crucial for dual specificity.
Arginine switches are not unique to engineered enzymes. Tyrosine
(aromatic)aminotransferase (TATase) is a well-characterized Fold
Type I enzyme that hasnatural specificity for the aromatic amino
acids tyrosine, phenylalanine, andtryptophan, as well as for the
dicarboxylic amino acids aspartate and glutamate.A number of
structures are now available of the Paracoccus denitrificansTATase
that clearly demonstrate the arginine switch (67, 68).
Crystallographic and modeling studies illuminated a similar
strategy employedby the GABA (69, 70) and ornithine (71)
aminotransferases, which react with both
Scheme 12 Schematic of the arginine switch in aminotransferases
that react withboth dicarboxylic and aromatic amino acids. The
�-carboxylate of glutamate (left)interacts closely with Arg292.
This residue reorients to point out of the active sitewhen aromatic
substrates bind (right). This movement allows the enzyme to
acceptboth types of substrates. Adapted from Reference 62.
401PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
�-amino acid substrates and the common substrate glutamate. GABA
ATase, likeAATase and TATase, binds the dicarboxylic acid substrate
via two conservedarginines. The carboxylate of the �-amino acid,
GABA, occupies the same positionas the �-carboxylate of glutamate,
thereby taking advantage of the similar distancebetween the amino
and carboxylate groups of GABA and the amino and �-carbox-ylate
groups of glutamate. The second arginine (equivalent to Arg386 of
AATase)moves to interact with a conserved glutamate near the active
site (Scheme 13).
HYDROGEN BOND NETWORKS Arginine switches, however, are not
ubiquitous,even among TATases. Early sequence alignments indicated
that a subgroup ofaminotransferases (designated I�) lack an Arg292
equivalent (72). The onlystructure available for a TATase of this
group is that of the unligandedPyrococcus horikoshii enzyme (73).
Modeling of the substrates in the active sitesuggests that this
enzyme binds glutamate via an extended hydrogen-bondingnetwork, as
has been observed in the AATase from this same organism (74)(Scheme
14). The absence of a positively charged residue in this TATase
makesit much easier to accommodate the uncharged substrates by
simple rearrange-ment of the hydrogen bond network. Moreover,
absence of the flexible arginineside chain allows this enzyme to
distinguish between glutamate and aspartate.The specificity ratio
(kcat/Km
Glu)/(kcat/KmAsp) for the P. horikoshii TATase is
3400 (73), compared to 0.27 for the Escherichia coli TATase
(75), a typicalmember of the I� family.
Scheme 13 Schematic of the arginine switch in GABA
aminotransferase. As in the caseof AATase, GABA ATase binds the
dicarboxylic acid substrate glutamate via twoconserved arginines.
In order to accommodate GABA, Arg445 moves away from thecofactor to
engage in a salt bridge with a nearby glutamate residue. Adapted
fromReferences 69 and 70.
402 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
The recently solved structures of the natural E. coli branched
chain amino acidaminotransferase (BCAT) (76) show that this enzyme
also takes advantage ofdifferent hydrogen bond interactions. BCAT
reacts with the branched chainaliphatic amino acids isoleucine,
leucine, and valine, as well as with glutamate.The hydrophobic
substrates and glutamate all bind in the same site, which is
ahydrophobic pocket consisting largely of aromatic residues. In
contrast to theTATases, there is no large-scale rearrangement of
the H-bond network toaccommodate the charged substrate. Instead,
the glutamate side chain, which isslightly longer than that of the
other substrates, extends into a pocket where fourresidues (Arg97,
Tyr31, Tyr129, and the backbone amide of Val109) hydrogenbond to
the carboxylate. The arginine in this case forms only a
monodentateinteraction rather than the bidentate H-bond network
seen in other enzymes, andit is extensively H-bonded to neighboring
residues, allowing it to remain inposition when the small
hydrophobic substrates are bound (Scheme 15).
EVOLUTION
Evolutionary relationships among PLP-dependent enzymes have been
exten-sively examined (25, 29, 72). The following discussion is
therefore limited torecent insights emanating from the increased
number of available structures.
Scheme 14 Dual substrate specificity can also be achieved by
hydrogen bondrearrangement. Pyrococcus horikoshii TATase binds the
�-carboxylate of glutamatevia a hydrogen bond network rather than
an arginine residue. Direct interactions aremade with a threonine
residue and a tightly bound water molecule. Modeling ofbound
tyrosine suggests that the hydrogen bond network rearranges, so
that thearomatic ring stacks against a nearby tyrosine residue, as
well as makes a hydrogenbond to the same threonine residue that is
involved in glutamate association. Adaptedfrom References 73 and
74.
403PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
The analyses of sequences and structures that led to the
categorization of PLPenzymes into the five recognized fold types
also indicated that the fold types areevolutionarily distinct (29).
The similarities of the cofactor binding sites thusprovide an
excellent example of convergent evolution. It is believed that
reactiontype generally evolved first within each fold type,
followed by narrowingsubstrate specificity (29). A number of
enzymes, however, group most closelywith those that catalyze
reactions of a different type, suggesting that theirreaction
type-specificity arose later in evolution. In these cases, the
change inreaction type can often be explained as a consequence of
altered substratespecificity. For example, enzymes catalyzing
�-elimination are found in manyevolutionary subgroups, often among
enzymes that catalyze transamination or�-elimination. Since
�-elimination of a good leaving group is a very facilereaction, it
is easy to imagine that the acquisition of improved binding of
asubstrate with a � leaving group could readily lead to a change in
the reactionspecificity to favor elimination. Another example of an
enzyme where a substratespecificity change effects a change in
reaction type is dialkylglycine decarbox-ylase (see above). DGD is
fundamentally an aminotransferase like most of itsclosest
evolutionary relatives, but it also catalyzes decarboxylation of
dialkylsubstrates that bind in a unique orientation.
Scheme 15 Dual substrate recognition by branched chain amino
acid aminotransferase.The substrate binding pocket is composed
primarily of aromatic residues, and the hydro-phobic substrate
isoleucine is surrounded by five of them, only three of which are
shown(Phe36, Tyr164, and Tyr31). The longer glutamate substrate
extends far enough to formhydrogen bonds with the hydroxyl groups
of two tyrosines and the guanidino group of anarginine residue.
Note that the orientation of the substrate C�-H bond is opposite
that foundin other fold types. Adapted from Reference 76.
404 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
L-Threonine-O-3-phosphate decarboxylase, an enzyme that is also
mostclosely related to aminotransferases, is unusual because the
change in reactiontype from transamination to decarboxylation
requires a different substrate ori-entation with respect to the
cofactor, and therefore mandates substantial rear-rangement of the
substrate binding mode. How this change was achievedevolutionarily
is evident in the fact that the closest evolutionary relative of
theenzyme is histidinol-phosphate aminotransferase, which has
evolved to bind aphosphate group in the position usually occupied
by the substrate carboxylate.One can imagine that from this
starting point (or something similar), it is easy toimprove binding
of threonine-phosphate in the same site. With the phosphate asan
anchor point in the usual carboxylate site, the carboxylate is
positioned forbond breakage (Scheme 16). It is not clear, however,
how decarboxylation-dependent transamination is averted, but
presumably the mechanism is similar tothose of other decarboxylases
and is related to the lack of a proton on the activesite lysine
after decarboxylation. Solvent exclusion can also prevent
hydrolysis ofthe ketimine intermediate formed by protonation at C4�
(78).
Possibly the most interesting group of PLP enzymes from an
evolutionarystandpoint is that of Fold Type IV (D-amino acid
aminotransferase (DAAT)family). This family is unique in containing
both a D-amino acid and an L-aminoacid aminotransferase (the
previously described BCAT). Since the active sitelysine can only
catalyze proton transfer on one face (79), DAAT and BCAT mustbind
their substrates in opposite orientations (Scheme 17), as evidenced
in thecrystal structures of these enzymes (76, 80). Either the
binding mode of the
Scheme 16 Possible evolutionary route to threonine phosphate
decarboxylase. Histidinolphosphate aminotransferase is quite
similar to Fold Type I amino acid aminotransferasesand must share a
common ancestral aminotransferase. However, it acquired
additionalaffinity for a phosphate group. Relatively minor changes
are required for that enzymeto accommodate threonine phosphate.
With the large phosphate group as an anchor,the carboxylate is
forced into the position occupied by the C� proton in the
relatedaminotransferases, thereby effecting a change in the
reaction type from transamination todecarboxylation.
405PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
substrate was reversed at some point during evolution, or the
enzymes share acommon ancestor with broad specificity.
MECHANISMS OF INHIBITION
The prominent role of PLP enzymes in metabolism has generated a
great deal ofinterest in the mechanisms of their inhibition.
Although they, like nearly allenzymes, are susceptible to simple
competitive inhibition, the catalytic versatilityof the cofactor
enhances their potential susceptibility to natural or
designedmechanism-based inhibitors. Because such inhibition is
often irreversible, it is ofmuch greater practical utility than
competitive inhibition (81). The inhibitedcomplexes are also
particularly useful for crystallographic studies, as they
oftenmimic substrate or reaction intermediate complexes. A large
number of inhibitorsof PLP enzymes have now been identified (for
more detailed reviews, see 6, 82),and they have been generally
grouped into three categories according to theirmode of
inactivation.
Noncovalent Inactivation
The simplest mechanism-based inhibitors of PLP enzymes are those
that formexceptionally stable complexes that often resemble normal
reaction intermedi-ates. Although the inhibitor is covalently bound
to the cofactor, the affinity forthe protein is through noncovalent
interactions. The combined affinity may bevery high. A recently
reported example is the stable ketimine intermediateformed in the
reaction of ACC synthase with L-aminoethoxyvinylglycine (83)(Scheme
18). The ketimine is an intermediate in the reaction catalyzed
by
Scheme 17 Reverse orientation of substrate binding by D-amino
acid aminotrans-ferase (DAAT) and branched chain amino acid
aminotransferase (BCAT). Theseclosely related enzymes are both in
Fold Type IV, where the active site lysine ispositioned on the re
face of the cofactor, opposite from its position in Fold Type I
andII enzymes. As a result, D-amino acids (shown bound to DAAT) are
bound with thecarboxylate pointing away from, and L-amino acids
(shown bound to BCAT) arebound with the carboxylate proximal to,
the cofactor phosphate group.
406 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
aminotransferases but not in the ACC synthase-catalyzed
elimination reaction;thus the enzyme is unable to catalyze its
hydrolysis. Of particular interest is thesubset of these inhibitors
whose stability is the result of enzyme-catalyzedaromatization. An
example is the inhibition of GABA ATase by
(S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid (84) (Scheme
19). Since the products ofthese reactions are not covalently
attached to the enzyme, the inhibited enzymescan often be
reactivated by dialysis in the presence of PLP, so that
thePLP-inhibitor species is replaced with fresh PLP (for example,
see 83), but thismode of reactivation does not generally occur on a
physiologically relevanttimescale.
Scheme 18 Inhibition of 1-aminocyclopropane-1-carboxylate
synthase by amino-ethoxyvinylglycine. The inhibitor reacts to form
a stable ketimine that is nothydrolyzed and remains tightly bound
to the enzyme. Dissociation by dialysisremoves the cofactor
together with the covalently bound inhibitor (83).
Scheme 19 Inhibition of �-aminobutyrate aminotransferase by
(S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid. The reaction
proceeds identically to the aminotransferasereaction up to the
formation of the ketimine intermediate. At this point,
deprotonation of a� carbon yields a very stable aromatic product
that does not react further and remains in theactive site (84).
407PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Activated Nucleophiles
A number of amino acids with good �-leaving groups, such as
�-chloroalanine,inhibit several types of PLP enzymes (6). The
general mechanism for thisreaction is by initial formation and
release of an enamine intermediate, which isa potent nucleophile
and can attack C4� of the cofactor (85) (Scheme 20).Inactivation is
irreversible, because the cofactor remains covalently bound to
theenzyme. An alternative proposed inactivation pathway (86) is by
direct Michaeladdition of an active site nucleophile to the
aminoacrylate aldimine. Freeaminoacrylate can also diffuse out of
the enzyme (as it does in natural �-elimination reactions), where
it spontaneously decomposes to pyruvate andammonia, leaving the
enzyme in an active form. Because of this possibility ofturnover as
well as inhibition, the effectiveness of these inhibitors is
oftenquantitated by the inactivation/turnover ratio.
Scheme 20 Mechanism of inactivation of a PLP-dependent enzyme by
�-chloroala-nine. The reaction follows the normal pathway to
�-elimination up to the formationof the aminoacrylate aldimine.
From there, the aminoacrylate may be released bytransaldimination
and may subsequently attack the cofactor nucleophilically.
Subse-quent hydrolysis of the imine yields the final inactivated
enzyme. A number of othermechanisms are possible, as noted in the
text.
408 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Activated Electrophiles
Inhibition can also result from rearrangement of the inhibitor
to generate anelectrophile that subsequently reacts irreversibly
with an active site nucleophile(often the active site lysine). Two
common types of inhibitors of this group areacetylenic compounds
such as propargylglycine, which reacts to form a highlyreactive
allene intermediate (87) (Scheme 21), and vinylic compounds such
asvinylglycine, which reacts to form an �,�-unsaturated imine
Michael acceptor(88) (Scheme 22). The reactivity of the potential
intermediates formed from theseinhibitors allows for alternative
reaction fates in addition to those shown. In thecase of
vinylglycine, for instance, an aminocrotonate aldimine, a
potentialMichael acceptor, can be formed from the quinonoid (89).
The aminocrotonatemay also be released by transaldimination, at
which point it can nucleophilicallyattack the cofactor in the
manner described above for aminoacrylate (90). A thirdpossible fate
is diffusion off the enzyme, where it decomposes spontaneously
to�-ketobutyrate and ammonia (91), leaving the enzyme in the active
PLP form.
Scheme 21 Mechanism of inactivation by propargylglycine. The
reaction parallelsa �-elimination mechanism through the formation
of the enamine intermediate. Atthis point, the acetylene moiety is
rearranged to form a highly reactive allene.Nucleophilic attack by
an active site residue results in covalent attachment to theenzyme.
The mechanism shown is slightly altered from that originally
proposed byAbeles & Walsh (87) to include a ketimine
intermediate in accord with the mostrecent proposal for the
mechanism of �-elimination (105).
409PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
The vinylglycine ketimine can also be hydrolyzed to release the
potentially toxicMichael acceptor 2-ketobut-3-enoic acid, leaving
the enzyme in the PMP form(92). Effective inhibitor design requires
limiting these possible alternativepathways.
Reversible Competitive Inhibition
Competitive inhibitors have proven to be exceptionally useful in
studies ofenzyme function and as unreactive substrate mimics in
crystallographic studies.There are two general classes of
competitive inhibitors—those that bind nonco-valently and those
that react reversibly with the enzyme to form an aldimine thatdoes
not react further. A classical example of the former is maleate,
whichinhibits AATase by binding in the aspartate site (6). This
association inducesclosure of the enzyme into the active form; thus
this inhibitor has proven usefulfor crystallographic studies of
this form of the enzyme.
The most common of the inhibitors that form unreactive aldimines
are�-methyl substrate analogs and amino-oxy or hydrazine analogs.
The closesimilarity of the �-methyl compounds to the substrates
makes them particularlyuseful for crystallography, and structures
of complexes of AATase and phos-phoserine aminotransferase with
�-methylaspartate and �-methylglutamate,respectively, have been
reported (93–95). Recent examples of the use of amino-oxy compounds
for structure determination are those of ACC synthase with
theamino-oxy analogue of SAM (96) (Scheme 23) and of ornithine
aminotransferasewith L-canaline, an analogue of ornithine (97)
(Scheme 23). The amino-oxyadducts with PLP are sufficiently stable
so that these compounds often need notbe substrate analogs.
Hydroxylamine itself binds to AATase to form a PLP-oxime whose Ki �
700 nM, a figure less than that for any of the
dicarboxylicinhibitor complexes (6). A final example of an
inhibitor that forms an unreactivealdimine is that of 1-aminoethyl
phosphonate (AlaP), which binds tightly to alanine
Scheme 22 Mechanism of inactivation by vinylglycine. The
reaction parallels an amino-transferase mechanism through the
formation of the ketimine intermediate. Michael addi-tion by an
active site nucleophile to the vinylglycine ketimine results in a
covalent adduct.
410 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
racemase (98). Although this molecule has a C� proton, it does
not undergodeprotonation. The stability of this complex enabled
determination of the enzymestructure with the inhibitor in the
active site, providing a mimic of the complex withthe natural
substrate (99).
The Challenge of Inhibitor Specificity
Although the variety of possible mechanisms of inhibition makes
it easy todesign effective inhibitors, specificity remains as a
major challenge for use invivo. Gabaculine (Scheme 23), for
example, is a potent inhibitor of GABAATase and also inhibits the
closely related ornithine aminotransferase (100),making it
unsuitable for pharmaceutical applications. One promising approach
isto incorporate the reactive functional groups described above in
structures thatare very close substrate analogs. An example is
�-vinylGABA (vigabatrin;Scheme 23), a specific inhibitor of GABA
ATase, which is used in the treatmentof epilepsy (reviewed in 101).
In this case, the vinylic group is appended to thenatural substrate
GABA to form a potential electrophile analogous to vinylgly-cine.
Another example is the ornithine aminotransferase (OAT) inhibitor
5-fluoromethylornithine (102) (Scheme 23). The fluoride ion is
susceptible to�-elimination, which generates an enamine capable of
nucleophilic attack on thecofactor in the manner described above
(71, 103), while the ornithine scaffoldprovides OAT
specificity.
Understanding mechanisms of inhibition is crucial not only to
enable the designof better inhibitors, but also to understand the
control of reaction mechanisms by thisimportant class of enzymes.
It is also of interest to ask how enzymes whose naturalreaction
pathways include reactive intermediates, such as the aminoacrylate
aldimineand vinylglycine ketimine, manage to avoid
inactivation.
CONCLUSIONS
The recent effusion of X-ray structures for PLP-dependent
enzymes—more thantwice as many were deposited in the protein data
bank between 1997 and 2001as in the preceding five years—has done
much to provide a visual framework in
Scheme 23 The structures of the inhibitors gabaculine,
vigabatrin, 5-fluoromethylorni-thine, canaline, and amino-oxy
SAM.
411PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
which the mechanistic concepts secured over the past
half-century can beinterpreted. The findings served to focus more
penetrating targeted mutagenesisexperiments and, taken together
with those from newer studies capitalizing upondirected evolution
methods, helped to elucidate some of the fundamental prin-ciples of
protein design.
We now have a good understanding of the mechanisms of dual
substraterecognition by aminotransferases and of how the fates of
the common C� anionare directed. Many questions remain unanswered,
particularly with regard to thecontrol of reaction pathways of both
natural substrates and mechanism-basedinhibitors. Of the four fold
types, only Fold Type I is well represented in thestructural
database, and many of the reaction types are only represented by
oneor two structures. We need additional structures of
underrepresented fold typesin order to direct further functional
studies. The understanding of the substrateand reaction
type-specificity of PLP enzymes is crucial for the design not only
ofspecific and medicinally useful inhibitors, but also of improved
protein-basedcatalysts.
ACKNOWLEDGMENT
The authors thank Susan Aitken and Kathryn McElroy for
critically reviewingthe manuscript.
The Annual Review of Biochemistry is online at
http://biochem.annualreviews.org
LITERATURE CITED
1. Heyl D, Luz E, Harris SA, Folkers K.1951. J. Am. Chem. Soc.
73:3430–33
2. Stryer L. 1995. Biochemistry. New York:Freeman
3. Voet D, Voet J. 1995. Biochemistry.New York: Wiley
4. Hayashi H. 1995. J. Biochem. 118:463–73
5. John RA. 1995. Biochim. Biophys. Acta1248:81–96
6. Christen P, Metzler DE, eds. 1985.Transaminases. New York:
Wiley
7. Miles EW. 2001. Chem. Rec. 1:140–518. Tai CH, Cook PF. 2001.
Acc. Chem. Res.
34:49–599. Ferreira GC, Gong J. 1995. J. Bioenerg.
Biomembr. 27:151–5910. Rao NA, Talwar R, Savithri HS. 2000.
Int. J. Biochem. Cell Biol. 32:405–16
11. Hutson S. 2001. Prog. Nucleic Acid Res.Mol. Biol.
70:175–206
12. He XM, Liu HW. 2002. Annu. Rev. Bio-chem. 71:701–54
13. Kleppner SR, Tobin AJ. 2001. EmergingTher. Targets
5:219–39
14. Snell K, Riches D. 1989. Cancer Lett.44:217–20
15. Wang CC. 1995. Annu. Rev. Pharmacol.Toxicol. 35:93–127
16. Mudd SH, Laster L, Finkelstein JD,Irreverre F. 1964. Science
143:1443–45
17. Kraus JP, Janosik M, Kozich V, MandellR, Shih V, et al.
1999. Hum. Mutat.13:362–75
18. Adams DO, Yang SF. 1979. Proc. Natl.Acad. Sci. USA
76:170–74
19. Honma M, Shimomura T. 1978. Agric.Biol. Chem. 42:1825–31
412 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
20. Keller JW, Baurick KB, Rutt GC,O’Malley MV, Sonafrank NL, et
al.1990. J. Biol. Chem. 265:5531–39
21. Watanabe Y, Shimura K. 1956. J. Bio-chem. 43:283–94
22. Rios A, Amyes TL, Richard JP. 2000.J. Am. Chem. Soc.
122:9373–85
23. Gallo KA, Knowles JR. 1993. Biochem-istry 32:3981–90
24. Alexander FW, Sandmeier E, Mehta PK,Christen P. 1994. Eur.
J. Biochem. 219:953–60
25. Jansonius JN. 1998. Curr. Opin. Struct.Biol. 8:759–69
26. Schneider G, Kack H, Lindqvist Y.2000. Struct. Fold. Des.
8:R1–6
27. Churchich JE, Moses U. 1981. J. Biol.Chem. 256:1101–4
28. Hennig M, Grimm B, Contestabile R,John RA, Jansonius JN.
1997. Proc.Natl. Acad. Sci. USA 94:4866–71
29. Mehta PK, Christen P. 2000. Adv. Enzy-mol. Relat. Areas Mol.
Biol. 74:129–84
30. Hyde CC, Ahmed SA, Padlan EA, MilesEW. 1988. J. Biol. Chem.
263:17857–71
31. Madison JT, Thompson JF. 1976. Bio-phys. Biochem. Res.
Commun. 71:684–91
32. Finkelstein JD, Kyle WE, Martin JJ, PickAM. 1975. Biophys.
Biochem. Res. Com-mun. 66:81–87
33. Gallagher DT, Gilliland GL, Xiao G,Zondlo J, Fisher KE, et
al. 1998. Struc-ture 6:465–75
34. Sugio S, Petsko GA, Manning JM, SodaK, Ringe D. 1995.
Biochemistry 34:9661–69
35. Kern AD, Oliveira MA, Coffino P,Hackert ML. 1999. Struct.
Fold. Des.7:567–81
36. Wolosker H, Blackshaw S, Snyder SH.1999. Proc. Natl. Acad.
Sci. USA96:13409–14
37. Contestabile R, Paiardini A, PascarellaS, di Salvo ML,
D’Aguanno S, Bossa F.2001. Eur. J. Biochem. 268:6508–25
38. Dunathan HC. 1966. Proc. Natl. Acad.Sci. USA 55:712–16
39. Kirsch JF, Eichele G, Ford GC, VincentMG, Jansonius JN, et
al. 1984. J. Mol.Biol. 174:497–525
40. Ploux O, Marquet A. 1996. Eur. J. Bio-chem. 236:301–8
41. Webster SP, Alexeev D, CampopianoDJ, Watt RM, Alexeeva M, et
al. 2000.Biochemistry 39:516–28
42. Sun S, Zabinski RF, Toney MD. 1998.Biochemistry
37:3865–75
43. Ford GC, Eichele G, Jansonius JN. 1980.Proc. Natl. Acad.
Sci. USA 77:2559–63
44. Borisov VV, Borisova SN, Sosfenov NI,Vainshtein BK. 1980.
Nature 284:189–90
45. Kuramitsu S, Inoue Y, Tanase S, MorinoY, Kagamiyama H. 1987.
Biochem. Bio-phys. Res. Commun. 146:416–21
46. Kochhar S, Finlayson WL, Kirsch JF,Christen P. 1987. J.
Biol. Chem. 262:11446–48
47. Toney MD, Kirsch JF. 1987. J. Biol.Chem. 262:12403–5
48. Cronin CN, Kirsch JF. 1988. Biochemis-try 27:4572–79
49. Hayashi H, Kuramitsu S, Inoue Y,Morino Y, Kagamiyama H.
1989. Bio-chem. Biophys. Res. Commun. 159:337–42
50. Kochhar S, Christen P. 1992. Eur. J. Bio-chem.
203:563–69
51. Shaw JP, Petsko GA, Ringe D. 1997.Biochemistry
36:1329–42
52. Albery WJ, Knowles JR. 1986. Biochem-istry 25:2572–77
53. Jencks WP. 1985. Chem. Rev. 85:511–2754. Paiardini A,
Contestabile R, D’Aguanno
S, Pascarella S, Bossa F. 2003. Biochim.Biophys. Acta
1647:214–19
55. Wolfenden R. 1974. Mol. Cell. Biochem.3:207–11
56. Schirch V, Shostak K, Zamora M,Guatam-Basak M. 1991. J.
Biol. Chem.266:759–64
57. Pasternak A, White A, Jeffery CJ,Medina N, Cahoon M, et al.
2001. Pro-tein Sci. 10:1331–42
413PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
58. Herschlag D. 1988. Bioorg. Chem.16:62–96
59. Goldberg JM, Kirsch JF. 1996. Biochem-istry 35:5280–91
60. Haruyama K, Nakai T, Miyahara I,Hirotsu K, Mizuguchi H, et
al. 2001.Biochemistry 40:4633–44
61. Onuffer JJ, Kirsch JF. 1995. Protein Sci.4:1750–57
62. Malashkevich VN, Onuffer JJ, KirschJF, Jansonius JN. 1995.
Nat. Struct. Biol.2:548–53
63. Yano T, Oue S, Kagamiyama H. 1998.Proc. Natl. Acad. Sci. USA
95:5511–15
64. Rothman SC, Kirsch JF. 2003. J. Mol.Biol. 327:593–608
65. Oue S, Okamoto A, Yano T, Kaga-miyama H. 1999. J. Biol.
Chem. 274:2344–49
66. Oue S, Okamoto A, Yano T, Kaga-miyama H. 2000. J. Biochem.
127:337–43
67. Okamoto A, Nakai Y, Hayashi H,Hirotsu K, Kagamiyama H. 1998.
J. Mol.Biol. 280:443–61
68. Okamoto A, Ishii S, Hirotsu K,Kagamiyama H. 1999.
Biochemistry38:1176–84
69. Toney MD, Pascarella S, De Biase D.1995. Protein Sci.
4:2366–74
70. Storici P, Capitani G, De Biase D, MoserM, John RA, et al.
1999. Biochemistry38:8628–34
71. Storici P, Capitani G, Muller R,Schirmer T, Jansonius JN.
1999. J. Mol.Biol. 285:297–309
72. Jensen RA, Gu W. 1996. J. Bacteriol.178:2161–71
73. Matsui I, Matsui E, Sakai Y, Kikuchi H,Kawarabayasi Y, et
al. 2000. J. Biol.Chem. 275:4871–79
74. Ura H, Harata K, Matsui I, Kuramitsu S.2001. J. Biochem.
129:173–78
75. Hayashi H, Inoue K, Nagata T,Kuramitsu S, Kagamiyama H.
1993. Bio-chemistry 32:12229–39
76. Goto M, Miyahara I, Hayashi H,
Kagamiyama H, Hirotsu K. 2003. Bio-chemistry 42:3725–33
77. Deleted in proof78. Eliot AC, Kirsch JF. 2003. Acc.
Chem.
Res. 36:757–6579. Soda K, Yoshimura T, Esaki N. 2001.
Chem. Rec. 1:373–8480. Peisach D, Chipman DM, Van Ophem
PW, Manning JM, Ringe D. 1998. Bio-chemistry 37:4958–67
81. Silverman RB. 1988. J. Enzyme Inhib.2:73–90
82. Nanavati SM, Silverman RB. 1989.J. Med. Chem. 32:2413–21
83. Capitani G, McCarthy DL, Gut H, Grut-ter MG, Kirsch JF.
2002. J. Biol. Chem.277:49735–42
84. Fu M, Nikolic D, Van Breemen RB, Sil-verman RB. 1999. J. Am.
Chem. Soc.121:7751–59
85. Likos JJ, Ueno H, Feldhaus RW, MetzlerDE. 1982. Biochemistry
21:4377–86
86. Kishore GM. 1984. J. Biol. Chem. 259:10669–74
87. Abeles RH, Walsh CT. 1973. J. Am.Chem. Soc. 95:6124–25
88. Rando RR. 1974. Biochemistry 13:3859–63
89. Soper TS, Manning JM, Marcotte PA,Walsh CT. 1977. J. Biol.
Chem. 252:1571–75
90. Nanavati SM, Silverman RB. 1991.J. Am. Chem. Soc.
113:9341–49
91. Miles EW. 1975. Biochem. Biophys. Res.Commun. 66:94–102
92. Choi S, Storici P, Schirmer M, Silver-man RB. 2002. J. Am.
Chem. Soc. 124:1620–24
93. McPhalen CA, Vincent MG, Picot D,Jansonius JN, Lesk AM,
Chothia C.1992. J. Mol. Biol. 227:197–213
94. Okamoto A, Higuchi T, Hirotsu K,Kuramitsu S, Kagamiyama H.
1994.J. Biochem. 116:95–107
95. Hester G, Stark W, Moser M, Kallen J,Markovic-Housley Z,
Jansonius JN.1999. J. Mol. Biol. 286:829–50
96. Capitani G, Eliot AC, Gut H, Khomutov
414 ELIOT y KIRSCH
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
RM, Kirsch JF, Grutter MG. 2003. Bio-chim. Biophys. Acta
1647:55–60
97. Shah SA, Shen BW, Brunger AT. 1997.Structure 5:1067–75
98. Badet B, Walsh C. 1985. Biochemistry24:1333–41
99. Stamper GF, Morollo AA, Ringe D,Stamper CG. 1998.
Biochemistry37:10438–45
100. Jung MJ, Seiler N. 1978. J. Biol. Chem.253:7431–39
101. Mumford JP, Cannon DJ. 1994. Epilep-sia 35:S25–28
102. Daune G, Gerhart F, Seiler N. 1988. Bio-chem. J.
253:481–88
103. Bolkenius FN, Knodgen B, Seiler N.1990. Biochem. J.
268:409–14
104. Sun S, Toney MD. 1999. Biochemistry38:4058–65
105. Brzovic P, Holbrook EL, Greene RC,Dunn MF. 1990.
Biochemistry 29:442–51
106. Livanova NB, Chebotareva NA, EroninaTB, Kurganov BI. 2002.
Biochemistry67:1089–98
107. Frey PA. 2001. Annu. Rev. Biochem.70:121–48
415PYRIDOXAL PHOSPHATE ENZYMES
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
Annual Review of Biochemistry Volume 73, 2004
CONTENTS
THE EXCITEMENT OF DISCOVERY, Alexander Rich 1
MOLECULAR MECHANISMS OF MAMMALIAN DNA REPAIR AND THE DNA DAMAGE
CHECKPOINTS, Aziz Sancar, Laura A. Lindsey-Boltz, Keziban
Ünsal-Kaçmaz, Stuart Linn 39
CYTOCHROME C -MEDIATED APOPTOSIS, Xuejun Jiang, Xiaodong Wang
87NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF HIGH-MOLECULAR-WEIGHT
PROTEINS, Vitali Tugarinov, Peter M. Hwang, Lewis E. Kay 107
INCORPORATION OF NONNATURAL AMINO ACIDS INTO PROTEINS, Tamara L.
Hendrickson, Valérie de Crécy-Lagard, Paul Schimmel 147
REGULATION OF TELOMERASE BY TELOMERIC PROTEINS, Agata
Smogorzewska, Titia de Lange 177CRAWLING TOWARD A UNIFIED MODEL OF
CELL MOBILITY: Spatial and Temporal Regulation of Actin Dynamics,
Susanne M. Rafelski, Julie A. Theriot 209
ATP-BINDING CASSETTE TRANSPORTERS IN BACTERIA, Amy L. Davidson,
Jue Chen 241
STRUCTURAL BASIS OF ION PUMPING BY CA-ATPASE OF THE SARCOPLASMIC
RETICULUM, Chikashi Toyoshima, Giuseppe Inesi 269DNA POLYMERASE ,
THE MITOCHONDRIAL REPLICASE, Laurie S. Kaguni 293
LYSOPHOSPHOLIPID RECEPTORS: Signaling and Biology, Isao Ishii,
Nobuyuki Fukushima, Xiaoqin Ye, Jerold Chun 321
PROTEIN MODIFICATION BY SUMO, Erica S. Johnson 355
PYRIDOXAL PHOSPHATE ENZYMES: Mechanistic, Structural, and
Evolutionary Considerations, Andrew C. Eliot, Jack F. Kirsch
383
THE SIR2 FAMILY OF PROTEIN DEACETYLASES, Gil Blander, Leonard
Guarente 417
INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS AS SIGNAL INTEGRATORS,
Randen L. Patterson, Darren Boehning, Solomon H. Snyder 437
STRUCTURE AND FUNCTION OF TOLC: The Bacterial Exit Duct for
Proteins and Drugs, Vassilis Koronakis, Jeyanthy Eswaran, Colin
Hughes 467
ROLE OF GLYCOSYLATION IN DEVELOPMENT, Robert S. Haltiwanger,
John B. Lowe 491
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.
-
STRUCTURAL INSIGHTS INTO THE SIGNAL RECOGNITION PARTICLE,
Jennifer A. Doudna, Robert T. Batey 539
PALMITOYLATION OF INTRACELLULAR SIGNALING PROTEINS: Regulation
and Function, Jessica E. Smotrys, Maurine E. Linder 559
FLAP ENDONUCLEASE 1: A Central Component of DNA Metabolism, Yuan
Liu, Hui-I Kao, Robert A. Bambara 589
EMERGING PRINCIPLES OF CONFORMATION-BASED PRION INHERITANCE,
Peter Chien, Jonathan S. Weissman, Angela H. DePace 617
THE MOLECULAR MECHANICS OF EUKARYOTIC TRANSLATION, Lee D. Kapp,
Jon R. Lorsch 657
MECHANICAL PROCESSES IN BIOCHEMISTRY, Carlos Bustamante, Yann R.
Chemla, Nancy R. Forde, David Izhaky 705
INTERMEDIATE FILAMENTS: Molecular Structure, Assembly Mechanism,
and Integration Into Functionally Distinct Intracellular Scaffolds,
Harald Herrmann, Ueli Aebi 749
DIRECTED EVOLUTION OF NUCLEIC ACID ENZYMES, Gerald F. Joyce
791
USING PROTEIN FOLDING RATES TO TEST PROTEIN FOLDING THEORIES,
Blake Gillespie, Kevin W. Plaxco 837
EUKARYOTIC mRNA DECAPPING, Jeff Coller, Roy Parker 861
NOVEL LIPID MODIFICATIONS OF SECRETED PROTEIN SIGNALS, Randall
K. Mann, Philip A. Beachy 891
RETURN OF THE GDI: The GoLoco Motif in Cell Division, Francis S.
Willard, Randall J. Kimple, David P. Siderovski 925
OPIOID RECEPTORS, Maria Waldhoer, Selena E. Bartlett, Jennifer
L. Whistler 953
STRUCTURAL ASPECTS OF LIGAND BINDING TO AND ELECTRON TRANSFER IN
BACTERIAL AND FUNGAL P450S, Olena Pylypenko, Ilme Schlichting
991
ROLES OF N-LINKED GLYCANS IN THE ENDOPLASMIC RETICULUM, Ari
Helenius, Markus Aebi 1019
ANALYZING CELLULAR BIOCHEMISTRY IN TERMS OF MOLECULAR NETWORKS,
Yu Xia, Haiyuan Yu, Ronald Jansen, Michael Seringhaus, Sarah
Baxter, Dov Greenbaum, Hongyu Zhao, Mark Gerstein 1051
Ann
u. R
ev. B
ioch
em. 2
004.
73:3
83-4
15. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Illin
ois
- U
rban
a C
ham
paig
n on
09/
27/1
1. F
or p
erso
nal u
se o
nly.