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Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103
3To whom correspondence should be addressed. E-mail: jakubows{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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KEY WORDS: • translational editing • S-nitroso-homocysteine • homocysteine thiolactonase • protein N-homocysteinylation • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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, 2
). The information
transfer involves matching amino acids (AA) with cognate tRNA according
to the rules of the genetic code. The chemical activation involves
formation of a high energy ester bond between the carboxyl group of an
AA and a hydroxyl of the 3'-terminal adenosine of tRNA, with an
aminoacyl adenylate as intermediate.
 | (1) |
 | (2) |
Aminoacyl adenylate formation (Equation 1)
is the least accurate
step in the tRNA aminoacylation pathway. Some AA [e.g., Met vs.
homocysteine (Hcy); Ile vs. Val and Hcy; Leu vs. Hcy; Val vs. Cys and
Thr; Ala vs. Gly; Lys vs. ornithine (Orn); or Thr vs. Ser] are so
similar that AARSs misactivate them at frequencies exceeding the
frequency of translational errors, forming AARS-bound noncognate
aminoacyl adenylates. Noncognate adenylates are directly or indirectly
destroyed by the editing function of an AARS (Table 1
). Editing can occur by two alternative pathways (3
):
pretransfer, by hydrolysis of the noncognate aminoacyl adenylates or
post-transfer, by the hydrolysis of the mischarged tRNA. Because of
the fast dissociation of aminoacyl-tRNA from AARS,
post-transfer editing can contribute only a factor of 
2 to
selectivity (4
, 5
). This may explain the more widespread
use of pretransfer editing pathways by AARSs. Overall, editing improves
the AA selectivity of an AARS by a factor > 100
(5
, 6
). Consequently, nonprotein AAs Hcy or Orn are not
transferred to tRNA. Some noncognate AAs are transferred to tRNA with
low efficiency (Table 1)
. For instance, IleRS promotes one misacylation
of tRNAIle with Val per 350,000 correct
acylations with Ile. ValRS promotes one misacylation of
tRNAVal with Ile and Thr per 5,000 and 350,000
correct acylations with Val, respectively (6
). LysRS
catalyzes one misacylation of tRNALys with Arg,
Thr, Met, Leu, Ala, Cys or Ser per 1,600, 16,000, 32,000, 132,000,
265,000, 560,000 or 750,000 correct acylations with Lys, respectively
(7
). Some AARS, such as TyrRS, CysRS (1
, 6
),
ArgRS (7
), AspRS and SerRS (9
) bind the
cognate AA so much more tightly than their competitors that they do not
need to edit. Overall, the accuracy of tRNA aminoacylation is greater
than the accuracy of subsequent steps of translation on ribosomes
(6
).
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Molecular basis of editing |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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adenosine
5'-monophosphate (AMP) or AA-tRNA to yield a free AA.
However, side chains of Hcy, homoserine (Hse) or Orn participate
directly in editing, which results in the formation of cyclic
forms of these AAs (Fig. 1
). For example, methionyl-tRNA synthetase (MetRS), IleRS
(3
) and LysRS (9
) misactivate Hcy and form an
AARS-bound Hcy adenylate (Equation 3)
.
|
| (3) |
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AMP intermediate
involves an intramolecular reaction in which the side chain thiolate of
Hcy displaces the AMP group form the carboxylate of the activated Hcy,
forming Hcy thiolactone as a product. The energy of the anhydride bond
of HcyAMP is conserved in an intramolecular thioester bond of
thiolactone. Consequently, thiolactone easily acylates free amino
groups of protein lysine residues (10
).
In similar reactions, Hse is converted to Hse lactone during editing by
LysRS, IleRS or ValRS; Orn is converted to Orn lactam during editing by
LysRS (6
, 7
, 9
) (Fig. 1)
.
A model of the synthetic/editing active site explains how MetRS directs
Met and Hcy to the synthetic and editing pathways, respectively
(11
, 12
). Two subsites are important for the AA selectivity
of MetRS. First, the specificity subsite preferentially binds the side
chain of the cognate substrate Met by hydrophobic and hydrogen bonding
interactions. The hydrophobic interactions involve side chains of
Tyr15, Trp253, Pro257 and Tyr260; Trp305 closes the bottom of the
hydrophobic Met-binding pocket but is not in contact with the
methyl group of the substrate Met (13
). Mutations of Tyr15
and Trp 305 residues affect Hcy/Met discrimination by the enzyme
(11
). The sulfur atom of the substrate Met makes two
hydrogen bonds, one with the hydroxyl of Tyr260 and the other with the
backbone amide of Leu13 (13
). Second, the
thiol-binding subsite preferentially binds the side chain of the
noncognate Hcy (12
). Similar synthetic/editing active site
model explains editing of Hcy by IleRS (14
).
In the synthetic pathway, the activated carboxyl group of Met reacts
with the 2'-hydroxyl of the terminal adenosine of
tRNAMet, yielding
Met-tRNAMet. In the editing pathway, the
activated carboxyl group of Hcy reacts with the sulfur of its side
chain, yielding Hcy thiolactone. Whether an AA completes the synthetic
or editing pathway is determined by the competition for its activated
carboxyl group between the side chain of the AA and the terminal
adenosine of tRNAMet. Met completes the synthetic
pathway because its side chain is firmly bound by hydrophobic and
hydrogen bonding in the specificity subsite, preventing the sulfur atom
of Met from competing with the 3'-terminal adenosine of
tRNAMet for the carboxyl carbon of Met. The side
chain of Hcy, missing the methyl group of Met, interacts with the
specificity subsite much more weakly than does the side chain of Met.
This allows the side chain of Hcy to also interact with the
thiol-binding subsite, which facilitates editing (Fig. 2
). This explains why Hcy is not transferred to tRNA but is cyclized to
Hcy thiolactone. Met can also enter the editing pathway when the thiol
subsite is occupied by a thiol mimicking the side chain of Hcy
(12
) (Fig. 3
).
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) can be accommodated in a single active site
(12
, 14
), tRNA-dependent editing of Val by IleRS and of
Thr by ValRS seem to involve a separate site for editing; this is known
as a double-sieve mechanism (1
). For example, a
polypeptide (CP1) derived from the nucleotide-binding fold of IleRS
exhibits a weak but specific hydrolytic activity toward
Val-tRNAIle (15
). It is not known,
however, whether CP1 can also hydrolyze ValAMP. Crystallographic
studies of IleRS reveal that Ile binds at the active site in the
nucleotide-binding fold domain (16
). In a complex of
IleRS with Val, one Val is bound to the same active site but a second
Val binds to the CP1 polypeptide. Mutation of AA residues in the second
Val binding site abolishes editing activity of IleRS and the mutant
IleRSs now mischarge tRNAIle with Val
(16
). A translocation of ValAMP from the synthetic to
the editing site, over a distance of 25 Å, is proposed to occur
(17
). This editing mechanism seems to be idiosyncratic in
that it is unlikely that other noncognate AA edited by IleRS, such as
Leu or Hcy (Table 1)
, can enter the editing site for valine. |
Decoding methionine codons by Hcy |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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)
(Fig. 4
). This in turn allows translational incorporation of
S-NO-Hcy into protein in the bacterium Escherichia
coli and in an in vitro rabbit reticulocyte system. Removal of the
nitroso group yields proteins containing Hcy at positions normally
occupied by methionine (18
). Translationally incorporated
Hcy is also present in cultured human endothelial cells (Table 2
). Thus, Hcy can gain access to the genetic code by
S-nitrosylation-mediated invasion of the
methionine-coding pathway.
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Aminoacylation of coenzyme A and synthesis of AA-Cys by AARS |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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, 14
, 19
, 20
). With CoA-SH and cysteine, which are
preferred thiol substrates, these reactions yield
aminoacyl-S-CoA thioesters and AA-Cys dipeptides,
respectively. The thiol aminoacylation reactions are catalyzed by other
tested class I (Arg-, Cys-, and ValRS) and class II AARSs (Asp-, Ser-,
and LysRS) (8
, 9
, 20
). Thus, CoA-SH/Cys-binding sites exist
in present-day AARSs, perhaps indicative of a vestigial
thiol-dependent peptide-synthesizing function, similar to that of
the present-day nonribosomal peptide synthesis by multienzyme
thiotemplate systems. In addition, both AARS and nonribosomal peptide
synthetases exhibit relaxed AA selectivity in the thioester bond
formation reaction, likely indicative of more primitive aminoacylation
systems (20
). |
Editing is part of the process of tRNA aminoacylation in vivo |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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–28
). Exogenous
Hcy is also metabolized to thiolactone by two other synthetases, IleRS
and LeuRS, at least in E. coli (23
). The
C-terminal domain of MetRS is important for the editing of
endogenous Hcy (28
). These findings show that editing is
part of the process of tRNA aminoacylation in all cell types and that
AAs are channeled from the biosynthetic pathway to the protein
biosynthetic apparatus (Fig. 5
).
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Hcy thiolactone and protein N-homocysteinylation in humans |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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) depends on the concentrations of Hcy (or the Hcy:Met ratio), folic
acid and high density lipoprotein (HDL), factors that have been
implicated in susceptibility to vascular disease in humans. Under
deregulated metabolic conditions (low folate, high Hcy), protein
N-homocysteinylation increases relative to that under normal
metabolic conditions (high folate, low Hcy) (25
, 27
, 29
).
Incorporation of Hcy into HUVEC proteins occurs by
post-translational (Hcy thiolactone-mediated) and translational
(most likely nitric oxide mediated) mechanisms (Table 2)
.
|
) and Hcy-N-protein are present in human plasma. In normal
human plasma, levels of Hcy thiolactone and Hcy-N-protein
represent 1.5% and 15%, respectively, of total Hcy levels
(Jakubowski, H., unpublished data). The levels of
Hcy-N-protein positively correlate with Hcy levels in plasma
(Fig. 8
).
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Human serum Hcy thiolactonase minimizes protein N-homocysteinylation |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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), the ability to detoxify the
thiolactone is essential for biological integrity. Indeed, Hcy
thiolactone is hydrolyzed to Hcy by a single specific enzyme, Hcy
thiolactonase, present in mammals but not in birds (30
).
Human serum thiolactonase, a 45-kD protein component of HDL, requires
calcium for activity and stability and is inhibited by penicillamine
(Ki = 0.2 mM) and isoleucine
(Ki = 2 mM). HTase also hydrolyzes the
organophosphate paraoxon and is identical with paraoxonase, a product
of the PON1 gene, implicated in human cardiovascular disease.
Thiolactonase actitivity is absent in mice lacking paraoxonase
(30
). Human PON1 has several genetic
polymorphisms, two of which result in AA substitutions at positions 55
and 192 in PON1 proteins. There is a strong association
between thiolactonase activity and PON1 genotype in human
populations (31
): high thiolactonase activity is
associated with L55 and R192 alleles, more frequent in blacks (0.82 L55
and 0.64 R192) than in whites (0.64 L55 and 0.39 R192); low
thiolactronase activity is associated with M55 and Q192 alleles, more
frequent in whites (0.36 M55 and 0.61 Q192) than in blacks (0.18 M55
and 0.36 Q192). The high activity form of thiolactonase affords better
protection against protein N-homocysteinylation than the low activity
form (31
). |
Implications for cardiovascular disease |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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). However,
it is not known why Hcy is harmful. A plausible hypothesis is that Hcy
is harmful because it can be incorporated into protein by
post-translational (thiolactone-mediated) and translational
(S-nitrosylation-mediated) mechanisms, which lead to protein
damage. These mechanisms are absolutely specific for Hcy because Hcy
thiolactone and S-NO-Hcy can only arise from Hcy. The levels
of Hcy thiolactone and Hcy-N-protein depend on the ratio of
Hcy to Met, the levels of folic acid, and HDL (27
),
factors linked to cardiovascular diseases. Variations in
HDL-associated thiolactonase affect protein homocysteinylation and,
therefore, may play an important role in Hcy-linked human
cardiovascular disease.
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FOOTNOTES |
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2 Supported by Grants MCB-9724929 and MCB-0089984
from the National Science Foundation, and Grant 22-02 from UMDNJ
Foundation.
4 Abbreviations used: AA, amino acid; AARS,
aminoacyl-tRNA synthetase; AMP, adenosine 5'-monophosphate; Hcy,
homocysteine; Hse, homoserine; HDL, high density lipoprotein; HUVEC,
human umbilical vein endothelial cells; MetRS, methionyl-tRNA
synthetase; Orn, ornithine.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMolecular basis of editingDecoding methionine codons by...Aminoacylation of coenzyme A...Editing is part of...Hcy thiolactone and protein...Human serum Hcy thiolactonase...Implications for cardiovascular...LITERATURE CITED |
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1. Fersht, A. (1999) Structure and Mechanism in Protein Science 1999:374-389 W. H. Freeman & Co New York, NY. .
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4. Lin, S. X., Baltzinger, M. & Remy, P. (1984) Fast Kinetic study of yeast phenylalanyl-tRNA synthetase: role of tRNA in the discrimination between tyrosine and phenylalanine. Biochemistry 23:4109-4116.[Medline]
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13. Serre, L., Verdon, G., Choinowski, T., Hervouet, N., Risler, J.-L. & Zelwer, C. (2001) How methionyl-tRNA synthetase creates its amino acid recognition pocket upon L-methionine binding. J. Mol. Biol. 306:863-876.[Medline]
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17. Silvian, L. F., Wang, J. & Steitz, T. A. (1999) Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and muciprocin. Science 285:1074-1077.
18. Jakubowski, H. (2000) Translational incorporation of S-nitroso-homocysteine into protein. J. Biol. Chem. 275:21813-21816.
19. Jakubowski, H. (1998) Aminoacylation of coenzyme A and pantetheine by aminoacyl-tRNA synthetases: possible link between non-coded and coded peptide synthesis. Biochemistry 37:5147-5153.[Medline]
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26. Jakubowski, H. & Goldman, E. (1993) Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells. FEBS Lett 317:593-598.
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