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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 Ia-hydrophobic amino acids (Ile, Leu, Val)-sulfur amino acids (Met and Cys)-Arg

-subclass Ib-charged amino acids (Glu, Lys) and derivative Gln

-subclass Ic-aromatic amino acids (Tyr and Trp)

-acylate the 2’-hydroxyl group of terminal adenosine of tRNA

Class II-10 members that possess a 7-stranded -sheet with flanking -helices

-subclass IIa-aliphatics (Ala, Pro)-polar (Ser, Thr, His) -Gly

-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

Chain 3TS1:_

Compound Tyrosyl-Transfer RNA Synthetase (E.C. 6.1.1.1) Complexed With Tyrosinyl Adenylate - Chain _

Type Protein

Molecular Weight 47290

Number of Residues

419

Number of Alpha 17 Content of Alpha 37.95

Number of Beta 6 Content of Beta 6.21

Sequence and secondary structure

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

G3:F30-8