Principles of Bioinorganic Chemistry - 2003 You should have your paper topic approved by Prof. Lippard this week, if you have not done so already (by 10/12.

Principles of Bioinorganic Chemistry - 2003

Lecture Date Lecture Topic Reading Problems1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/11 (Th) Metallochaperones; Metal Folding, X-linkingCh. 7 Ch. 34 9/16 (Tu) Zinc Fingers; Metal Folding; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Cisplatin; Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) ET Units; Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) ET; Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 10/ 7 (Tu) Model Complexes for MetallohydrolasesCh. 10 Ch. 89 10/9 (Th) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 910 10/10 (Fr)* O2 Activation, Hydroxylation: MMO, P-450, R2Ch. 11 Ch. 1011 10/14 (Tu) Model Chemistry for O2 Carriers/Activators Ch. 12 Ch. 1112 10/16 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1213 10/21 (Tu) Metalloneurochemistry/Medicinal Inorg. Chem.14 10/23 (Th) Term Examination

*Makeup class, 8:30 – 10 AM; room to be announced

You should have your paper topic approved by Prof. Lippard this week, if you have not done so already (by 10/12 please). The oral presentations will be held in research conference style at MIT's Endicott House estate

in Dedham, MA, on Saturday, October 18. WEB SITE: web.mit.edu/5.062/www/

Hydrolytic Enzymes, Zinc and other Metal Ions

PRINCIPLES:

•M(OH)n+ centers supply OH- at pH 7 by lowering water pKa

•Mn+ serves as general Lewis acid, activating substrates•Rate acceleration occurs by internal attack within coord. sphere•Protein side chains greatly assist assembly of transition state•Carboxylate shifts can occur, especially at dimetallic centers•Electrostatic interactions predominate•Non-redox active metal ions often but not universally used

Illustrating the Principles:

•Carboxypeptidase, carbonic anhydrase - delivering hydroxide•Alcohol dehydrogenase: an oxidoreductase•Dimetallic metallohydrolases: are two metals better than one?

Carboxypeptidase A: A Hydrolytic Zinc Enzyme

Reaction catalyzed:

R–CH–C(O)–NH–R’

NH2R’’

R–CH–CO2- + +NH3–R’

NH2R’’Cleaves C-terminal peptide bonds; prefers aromatic residues.

Active site contains a single catalytic zinc, essential for activity. The glutamate can undergo a carboxylate shift. Thermolysin has a similar active site; it is an endopeptidase.

Carboxypeptidase A structure with the inhibitor glycyl-L-tyrosine bound at the active site. Note hydrogen bonds to key residues in the active site that position the substrate moiety for bond scission.

Catalytic Mechanism for Carboxypeptidase ASummary of events:1. Substrate binds; orients by the terminal carboxylate.2. Deprotonate bound H2O.3. Polarize scissile bond by Arg127.4. Bound OH- attacks peptide C(O).5. Form tetrahedral transition state.6.Lose 2 peptide fragments and recycle the enzyme.

Principles illustrated:1. Zinc serves as template.2.Metal supplies cleaving reagent, OH-, and organizes key groups.3. Chemistry achieved at neutral pH! Kcat ~ 100 s-1 .

Carbonic Anhydrase, the First Known Zn Enzyme

Reaction catalyzed:

CO2 + H2O H2CO3 ~ 106 s-1

Note: Rate 10-2 s-1 at pH 7; kf 106 s-1 in active site.Paradox: The reverse reaction is diffusion controlled, with kr ~ 1011 M-1 s-1

Thus kf ≤ 104 s-1. So how can the turnover be 106 s-1 ? Answer: Facilitated diffusion of protons by buffer components bound to the enzyme.

PZn(OH2)2+ PZn(OH)+ + H+Keq = 10-7 M = kf/kr

Carbonic Anhydrase

Possible Carbonic Anhydrase Mechanism

Alcohol Dehydrogenase, an Oxidoreductase

Reaction catalyzed:RCH2 OH + NAD+ RCHO + NADH + H+

Enzyme contains two 40 kDa polypeptides, each with 2 Zn2+centers in separate domains. One zinc is structural, the other catalytic.

Catalytic zinc is 20 Å from the surface, near the nicotinamide binding region. This center is not required for NAD + cofactor binding. Alcohol substate DO require zinc and bind directly to the metal center, displacing the coordinated water.

Schematic Diagram

NAD+ binding to the active site of LADH, with specific, well-positioned amino acid side chains holding it in place. Ethanol is shown bound to the zinc, displacing water. The system is set to undergo catalysis.

Hydride Transfer Mechanism

N

H2N O

R' +

OZn

H

RH

H2O

N

H2N O

R' ..

H

H

+ RCHO +

H2OZn

Note hydride transfers from -C of alcohol to nicotinamide ring.

Dinuclear Metalloenzymes

Peptide hydrolases: Methionine aminopeptidase (Zn2 or Co2)Leucine aminopeptidase (Zn2)

Phosphoester hydrolases:Ser/Thr phosphatases (Fe/Zn or Fe/Fe)Alkaline phosphatase (Zn2)Nuclease P1 (Zn2)Inositol Monophosphatase (Mg2)RNase (Mn2 and Mg2)DNA polymerase I (Mg2)

Other metallohydrolases:Arginase (Mn2, Co2)Urease (Ni2)β- (Lactamase Zn2)

(Xylose isomerase Mg2):Isomerase

- Redox active dinuclear:Metalloenzymes

(Methane monooxygenase Fe2) (Tyrosinase Cu2)

(Catalase Mn2)

Alkaline Phosphatase; a Dizinc(II) Center Activates the Substrate

1. The substrate binds to the dizinc center; a nearby Arg also helps activate it.2. A serine hydroxyl group attack the phosphoryl group, cleaving the ester. The phosphate is transferred to the enzyme, forming a phosphoryl-serine residue.3. Hydrolysis of this phosphate ester by a zinc-bound hydroxide com-pletes the catalytic cycle.

This mechanism is supported by studies with chiral phosphate esters (ROP18O17O16O)2-; there is no net change in chirality at phoshorus.

1.

2.

3.

pKa Values of Metal-Bound Water for CommonMetal Ions in Aqueous Solution

pH1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fe3+-OH

Cu2+-OH

Zn2+-OH

Co2+-OH

Ni2+-OH

Mn2+-OH

Mg2+-OH

M OH2 M OH H+pKa

pKa

Barnum, D. W. Inorg. Chem. 1983, 22, 2297.

+

Dimetallics can move the value into the physiological range near pH 7

MB

OC

O

MA

R

N S

MB

OC

O

MA

R

S

MB

OC

O

MA

R

N S

MB

OC

O

MA

R

N SMB

OC

O

MA

R

N S

Modes of Substrate (S) Attack by an Activated Nucleophile (N) at a Carboxylate-Bridged Dimetallic Center

A EB C D

N:: :: :

Advantages of Carboxylate-Bridged Dimetallic Centersin Chemistry and Biology

O

C

O-

H2C

C

H

H3N COO-

O

C

O

Carbonate

CH2H2C

C

H

H3N COO-

Biologically available carboxylates:

O

C

O

OH

O

C

O-

NHH2C

C

H

H3N COO-

Aspartate (Asp) D

Carboxylate Ligation in Metalloproteins and the Carboxylate Shift

Glutamate (Glu) E

CH2

H2C

-

Lys* Carbamate

-

CH2

Carbonate is encountered in transferrinLys* is found in urease and rubisco

C

O

O

R

R

CO

NM

OOC

O R

N

C

O

O

R

R

CO

N

MO O

COR

NM

O

OR

M M

O O

R

M M

M M

O

O

R

Rardin, Bino, Poganiuch, Tolman, Liu, LippardAngew. Chem. Int. Ed. Engl., 102, 812 (1990)

Chemistry Leading to the Definition of the Carboxylate Shift

[M3II(O2CCH3)6(BIPhMe)2]

M = Mn, FeM(O2CCH3)2 + BIPhMe methanol

"This movement, which we term 'the carboxylate shift,'may be of general importance in the active site structuresof metalloproteins."

Note syn/anti lonepair traversals

The Expanded Carboxylate Shift

M

O

M

OCR

M

OC

O

M

R

MO

M

CR O

O

M

M

OO

M

M

CR O

O

M

C

C

R

O R

M

OM

CR

O

MO

M

OOC

R

M

O

M

CR O

CR O

M MM

The Active Sites of Selected Dinuclear MetalloenzymesCatalyzing Hydrolysis of Biological Substrates

FeO

Zn

HO

H2O

O

H2O

NN

N

O

HN

NH

NH

His92

His281

His199

OAsp90

O

Asp118

H2N

Asn150

Calcineurin

Zn Zn

HO

ON

N

N

O

HN

NH

NH

His6

His60

His116

O

Asp45

OO

Asp122

NH2

Trp1

Nuclease P1

Zn Zn

HO

O

N

N

O

HN

NH

His97

His156

O

Glu152

OO

Asp117

O

Aminopeptidase

Asp179

Zn Zn

HO

N

N

N

O

HN

NH

NH

His230

His57

His55

O

Asp301

OO

N

Phosphotriesterase

NHN

His201

Lys169

H

+H3N NH

NH2

CO2-

NH2+

+H3N NH3+

CO2- H2N NH2

O+

L-Ornithine

H2O +

L-Arginine Urea

Structure and Chemistry of Arginase

Mn2+ O

Mn2+

O

O

O

Asp-232

O O

Asp-124

O

O

Asp

OAsp-128

NNH

His

NHN

His

H

Active Site of Arginase

NH

NH2

NH2

O

O

Mn2+ OMn2+

NH

NH2

NH2

O

O

OMn2+ Mn2+

H

NH2

O NH2

NH2

O

OMn2+

Mn2+

Mn2+

H2O

Mn2+

III

L-Ornithine

II

IV

H2O

Urea

H+

L-Arginine

Postulated Catalytic Mechanism for Arginase

I O OO

OH

OO

Asp 124

Asp 128

Christianson, 1996

Principles illustrated: the dimetallic affords hydroxide; the substrate is positioned by residues in the active site; the dimetallic stabilizes the urea leaving group; redox inactive metal; electrostatics

The Dinickel(II) Metalloenzyme Urease

History of Urease

1926, Sumner crystallizes urease

1975, Blakeley and Zerner discover that urease is a dinickel enzyme

1995, Hausinger and Karplus determine X-ray structure; unusual active site

Ni Ni

N

N

N

N

N

NN

N

N

OO

O

O

O

Urea Hydrolysis

H2N

O

NH2 H2N

O

OH

H2Ourease + NH3

NH3 + H2O

N

NH

N

HN

NHN

NHN

Ni1O O

Ni2

HN

Lys220*

OH(2)OH2 O

H2OO Asp363

His137

His139

Figure 1. Barrios & Lippard

NNH

N

HN

NHN

NHNNi1

O O

Ni2

HNLys220*

OO ONH2

O Asp363

His137

His139N

NH

N

HN

NHN

NHNNi1

O O

Ni2

HNLys220*

SHO O

O Asp363

His137

His139

P

NH2His249

His275His275

His249

His275

His249

Native and Inhibited Urease from B. Pasteurii

Benini et. al. Structure 1999, 7, 205-216.Benini et. al. JBIC 1998, 3, 268-273.

Native urease, 2.0 Å resolution

β- , Mercaptoethanol inhibited urease1.65 Å resolution

, 2.0 DAP inhibited urease Å resolution

NiNi

OH

NiNi

OH O

NH2H2N

NiNi

O O

NH2

O

NH2H2N

Proposed Mechanism of Urea Hydrolysis

O

NH2H

O

NH2

O

NHCH3H2N

O

NHOHH2N

O

NHOHHOHNOther urease substrates:

H2O

CO2 + (2)NH3

NiNi

OH

NiNi

OH O

NH2H2N

NiNi

OH2

O

NH2H2N

O

C

N

Alternative Mechanism of Urea Hydrolysis

H2O

CO2 + (2)NH3

PZn(OH2)2+ PZn(OH)+ + H+Keq = 10-7M = kf/kr

Metallo-β-lactamases, an Emerging Clinical Problem

Zn1

H162

H101

H99

HO

Zn2OH2

C181

D103

H223Zn1

H149

H86

H88

Zn2

H210

C168

OH

OO

D90

H2OZn1

H86

H84

H160

HO

Zn2H225

H2O

D88

H89

S185

N

S

C

OCOOH

R'

R" HN

SCO

COOH

R"

R'

OH

Bacillus cereus Stenotrophomonas maltophiliao

Zn Zn, 3.4 Å...Zn Zn, 3.5 and 4.4 Å... Zn Zn, 3.5 Å...

H2OReaction Catalyzed:

Active Sites:

Bacteroides fragilis

Zn2

Wat2

C181

Wat1

Zn1

H99

H101

H162

D103

H223

N.O. Concha, B.A. Rasmussen, K. Bush, O. Herzberg (1996), Structure 4, 823-836

β-Lactamase from Bacteroides fragilis

Active Site of a β- Lactam Antibiotic , -1 -Resistance Enzyme IMP Metalloβ-lactamase( , ., 1999Fitzgerald et al )

Zn ZnO

N

N

N

HN

HN

NH

His206

O

O

Asp86

His145

His84

N

NH

His82

H

S

Cys164

Zn Zn

N

S

O

O

O

R1

R2

NH

S

OH

O

O

R1

R2OH

HO

O O

Possible Mechanism for Metallo-β-lactamases

S

ONH

N

S

NO2

NO2

COO-O

SNH

O

O

OH HN

S

COO-

NO2

NO2

400 nm 496 nm

1

4

5 6

910

12

H2O

nitrocefin: a substrate for investigating the mechanism

Zn Zn

N-

S

O

O

O-

R1

R2

-O

O O

blue intermediate

•Both mono- and dimetallic centers lower the pKa value of bound water, allowing hydroxide to be delivered at pH 7.

•Coordination of the leaving group portion of the substrate to a metal ion activates the substrate for nucleophilic attack.

•Residues not coordinated but in the second coordination sphere can participate directly (serine in phophatases) or indirectly (arginine in alcohol dehydrogenase) in substrate attack, orientation, and/or activation.

•Carboxylate shifts facilitate substrate binding, activation.

•Redox inactive metal ions (Zn2+, Ni2+, Mn 2+, Co2+) preferred.

Summary - Points to Remember