1 Lecture #21: Aminoacyl tRNA Synthetases: The Ancient Enzyme (E.C.# 6.1.1.1) -RS are ancient enzymes over 3.5 billion years old -evolution/development closely connected with genetic code -RS enzymes are at the centre of research on the origin of life Reaction: Amino acid + tRNA + ATP aminoacyl—tRNA + AMP + PPi Classification: -at least one aminoacyl tRNA synthetase for each amino acid -grouped into two classes: -Class I and II -each class divided into three subclasses a, b, and c -each class originated from a single domain ancestor De Pouplana & Schimmel 2001 TiBS 26 , 591- 596.
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1 Lecture #21: Aminoacyl tRNA Synthetases: The Ancient Enzyme (E.C.# 6.1.1.1) -RS are ancient enzymes over 3.5 billion years old -evolution/development.
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Lecture #21: Aminoacyl tRNA Synthetases: The Ancient Enzyme (E.C.# 6.1.1.1)
-RS are ancient enzymes over 3.5 billion years old-evolution/development closely connected with genetic code-RS enzymes are at the centre of research on the origin of life
Reaction: Amino acid + tRNA + ATP aminoacyl—tRNA + AMP + PPi
Classification:-at least one aminoacyl tRNA synthetase for each amino acid-grouped into two classes:
-Class I and II-each class divided into three subclasses a, b, and c-each class originated from a single domain ancestor
De Pouplana & Schimmel 2001
TiBS 26, 591-596.
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Class I-11 enzymes with active site that has Rossmann fold (parallel -sheet domain)
-subclass IIb-charged amino acids (Asp, Lys) and derivative Asn
-subclass IIc-aromatic amino acid (Phe)
-acylate the 3’-hydroxyl group of terminal adenosine of tRNA
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Type I: Gln-tRNA Synthase from
E. coli
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Type II: Asp-tRNA Synthase from yeast
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-wide variety of structural types, especially in eukaryotes-, 2, 4, and 22 with total Mr values ranging from 50,000 – 300,000-subunit Mr values are larger in eukaryotes and involves an N-terminal extension (50 – 300 amino acids)
V&V:T32-4
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ReactionL4:F27-14
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Tyrosyl tRNA Synthetase
-X-ray crystallographic and protein engineering studies have provided insight into the catalytic mechanism of tyrosyl-tRNA synthase, a Class I dimer of 47 kDa subunits
-in centre of each subunit is a 6-stranded -sheet structure with 5 longer helices and several shorter ones
-a number of complexes have been examined by x-ray crystallography involving AMP, ATP, tyrosine, and tyrosyl-AMP (highly stable)
-amino terminal 320 residues are needed for the activation reaction
-carboxy terminal 99 residues participate in the binding of tRNA and the formation of the tyrosyl-tRNA
-activated intermediate is stable in the absence of matching tRNA and is bound to enzyme with 12 H-bonds
Tyr-tRNA synthetase with tyrosine-adenylate bound in active site
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Overview
Chains Residues Mol. Weight [D] Chain Type
3TS1:_ 419 47290 Protein
Download all chains in FASTA format
Secondary Structure Elements given below are documented in the Help Section
1 MDLLAELQWR GLVNQTTDED GLRKLLNEER VTLYCGFDPT ADSLHIGHLA HHHHHHHH T SEES HH HHHHHHHHS EEEEEE S SSS BTTTHH 51 TILTMRRFQQ AGHRPIALVG GATGLIGDPS GKKSERTLNA KETVEAWSAR HHHHHHHHHH TT EEEEEE TTTTTT T T SS HHHHHHHHHH 101 IKEQLGRFLD FEADGNPAKI KNNYDWIGPL DVITFLRDVG KHFSVNYMMA HHHHHHHHS SS SSS EE EETHHHHTT HHHHHHHTG GGTTHHHHTT 151 KESVQSRIET GISFTEFSYM MLQAYDFLRL YETEGCRLQI GGSDQWGNIT SHHHHTTTTT HHHHTHH HHHHHHHHHH HHHH EEE E GGGHHHHH 201 AGLELIRKTK GEARAFGLTI PLVTKADGTK FGKTESGTIW LDKEKTSPYE HHHHHHHHHH EEEE SSSS TT SS B SSTTTTTHHH 251 FYQFWINTDD RDVIRYLKYF TFLSKEEIEA LEQELREAPE KRAAQKTLAE HHHHHHTTTH HHHTHHHHHH HHHHHH HHHHHHHTTT TTHHHHHHHH 301 EVTKLVHGEE ALRQAIRISE ALFSGDIANL TAAEIEQGFK DVPSFVHEGG HHHHHHHTHH HHHHHHHH 351 DVPLVELLVS AGISPSKRQA REDIQNGAIY VNGERLQDVG AILTAEHRLE 401 GRFTVIRRGK KKYYLIRYA
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H-bonds and Specificity
-side chains implicated in H-bonds have systematically been replaced by non-H-bonding residues (eg., Cys 35 Gly, Tyr 34 Phe)
-side chains responsible for specificity of the enzyme for tyrosine as opposed to phenylalanine are: Tyr 34 and Asp 176
-in ribose binding site: Cys 35, Thr 51, and His 48
-Cys 35 is conserved in bacterial tyrosyl-tRNA synthases but replacement resulted in an enzyme with 30% of wild-type activity
-results of mutagenesis showed that the different types of H-bonds made different contributions to the binding energy
-mutation of an uncharged side chain (Tyr 169) that forms a hydrogen bond to a charged group on the substrate (the -amino group) weakens the binding by 15.5 kJ/mol
-mutation of a side chain (Tyr 34) that forms an H-bond to an uncharged group (the phenolic OH group of tyrosyl-AMP) weakens the binding by only 2.2 kJ/mol
-Thr 51 forms an unfavorable H-bond with the ribose of tyrosyl AMP; it could form a stronger H-bond with water promoting the dissociation of the tyrosyl-AMP complex
-mutation of Thr 51 to Pro or Ala improved the kcat by 50- and 2-fold, respectively
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S4: F34-9
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G3:F30-7a
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Catalysis-reaction proceeds by an in-line displacement mechanism where the tyrosyl carboxylate is the attacking nucleophile and the pyrophosphate is the leaving group
- phosphorus atom in the transition state is pentavalent and the geometry of this state is trigonal bipyramidal (cf. RNAse A)
-model for transition state includes the H-bonding of the -phosphate group to the side chains of Thr 40 and His 45
-double mutant, T40A and H45A, has a decreased kcat of 3.6 x 106 fold but binding affinity of the enzymes for ATP and tyrosine were unalteredshows Thr 40 and His 45 important for catalysis but not substrate binding
-these residues likely interact with the phosphate group in the transition state but not in the initial enzyme-substrate complex
-selective binding believed to be triggered by the large shift in position of the pyrophosphate unit accompanying the tetrahedral to bipyramidal geometry change
-classic instance that “the essence of catalysis is the selective stabilization of the transition state”
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What are the catalytic residues?
-perhaps there are none because the carboxylate group of Tyr is an intrinsically effective nucleophile, ATP is already activated, and Mg2+--PPi is a good leaving group
-enzyme may simply accelerate the reaction by a factor of 4 x 104 by bringing Tyr and ATP together and it may gain another factor of 3 x 105 mainly by binding phosphate in the transition state
-since ATP, amino acid, and pyrophosphate can each bind to the enzyme separately, the reaction is random-order ternary type
-in most cases the rate of the first reaction is 10 – 100 times the rate of the second reactions, but in some enzymes the rates are nearly equal
S4:F34-10
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Editing/Proofreading by Aminoacyl tRNA Synthetases
-aminoacyl tRNA synthetases are highly selective in their recognition of both the amino acid to be activated and the prospective tRNA acceptor
-tRNA molecules that accept different amino acids have different base sequences so they can readily be distinguished by their synthetases
How do these enzymes discriminate between Ile and Val?
-extra methylene group in Ile provides additional binding energy of –12 kJ/mol which favours the activation of Ile by isoleucine tRNA synthetase by a factor of 200
-however, concentration of Val in vivo is 5 times that of Ile Val should mistakenly be incorporated 1 in 40 times
-observed frequency is 1 in 3000 times editing function
-mistakenly activated Val is not transferred to tRNA specific for Ile
-this tRNA promotes the hydrolysis of Val—AMP and prevents the erroneous incorporation into proteins
-this hydrolysis frees the synthetase to activate and transfer Ile, the correct amino acid
-the enzyme avoids hydrolyzing the Ile-AMP because the hydrolytic site is just large enough to accommodate Val—AMP but too small to allow the entry of Ile-AMP
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S4:F34-12
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What about amino acids that are nearly identical in size (Val and Thr)?
-the aminoacyl tRNA synthetase for Val contains two adjacent catalytic sites, one for the acylation of tRNA and the other for the hydrolysis of incorrectly acylated tRNA
-Val is preferred over Thr in the acylation reaction because the acylation site is more hydrophobic
-threonyl tRNA is hydrolyzed more rapidly because the hydrolysis site is more hydrophilic
-the synthetase for Val does most of the editing at the level of the aminoacyl-tRNA whereas the one for Ile does so at the level of the aminoacyl-AMP-most aminoacyl tRNA synthetases contain hydrolytic sites in addition to acylation sites
-complementary pairs of sites function as a double sieve to assure high fidelity
-the acylation site rejects amino acids that are larger than the correct one whereas the hydrolytic site destroys activated intermediates that are smaller than the correct species
-hydrolytic proofreading is essential to the fidelity of many aminoacyl tRNA synthetases
-some synthetases do not require editing functions because the binding of other amino acids is much weaker eg., tyrosyl-tRNA synthetases bind Tyr 104 x stronger than Phe
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Aminoacyl Synthetase Recognition of tRNA
-some synthetases recognize their tRNA partner based on the anticodontRNAAla is recognized at the 3:70 position in the 3’ acceptor stem of this 76-nucleotide molecule
-tRNACys differs from tRNAAla at 40 positions and contains a C-G basepair at the 3:70 position
-when the C-G basepair is changed to G-U at the 3:70 position of the tRNACys then alanyl-tRNA synthetase recognizes it as though it were tRNAAla
-a microhelix containing 24 of the 76 nucleotides of the native tRNA is specifically recognized by alanyl-tRNA synthetase