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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 &
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Schimmel 2001
TiBS 26, 591-596.
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 22 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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Reaction
L4: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 tyrosineadenylate 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:_
Tyrosyl-Transfer RNA Synthetase (E.C. 6.1.1.1) Complexed With Tyrosinyl
Adenylate - Chain _
Type
Protein
Molecular Weight 47290
Number of
419
Residues
Number of Alpha 17
Content of Alpha
37.95
Number of Beta 6
Content of Beta
6.21
Compound
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-Hbonding 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 unaltered
shows 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 randomorder 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
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S4:F34-10
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 IleAMP
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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 aminoacyltRNA 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 anticodon
tRNAAla 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
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