Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

Lily Q Dong, Feng Liu, Alan M. Myers, Herbert J. Fromm

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Abstract

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [714C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 × 104. On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.

Original languageEnglish (US)
Pages (from-to)12228-12233
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number19
StatePublished - 1991
Externally publishedYes

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Adenylosuccinate Synthase
Mutagenesis
Chemical modification
Site-Directed Mutagenesis
Inosine Monophosphate
Escherichia coli
Phenylglyoxal
Arginine
Guanosine Triphosphate
Binding Sites
Substrates
Enzymes
Aspartic Acid
Enzyme inhibition
Enzyme activity
Leucine
Stoichiometry
Protein Sequence Analysis
Catalysis
Rate constants

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis",
abstract = "Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [714C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 × 104. On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.",
author = "Dong, {Lily Q} and Feng Liu and Myers, {Alan M.} and Fromm, {Herbert J.}",
year = "1991",
language = "English (US)",
volume = "266",
pages = "12228--12233",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

AU - Dong, Lily Q

AU - Liu, Feng

AU - Myers, Alan M.

AU - Fromm, Herbert J.

PY - 1991

Y1 - 1991

N2 - Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [714C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 × 104. On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.

AB - Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [714C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 × 104. On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.

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