Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies

Jae Hoon Shim, Stephen Benkovic

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E., Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281,485-499], permitting a more exact formulation of the probable catalytic mechanism.

Original languageEnglish (US)
Pages (from-to)10024-10031
Number of pages8
JournalBiochemistry
Volume38
Issue number31
DOIs
StatePublished - Aug 3 1999

Fingerprint

Phosphoribosylglycinamide Formyltransferase
Mutagenesis
Site-Directed Mutagenesis
Escherichia coli
Catalysis
pH effects
Apoenzymes
Catalytic Domain
Kinetics
Deuterium
Kinetic parameters
Isotopes
Ionization
Salts
Amino Acids
X rays
Catalysts
Acids
Geometry
Enzyme Inhibitors

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{d0f11988cb5445caacd313bcf8ba31aa,
title = "Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies",
abstract = "Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E., Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281,485-499], permitting a more exact formulation of the probable catalytic mechanism.",
author = "Shim, {Jae Hoon} and Stephen Benkovic",
year = "1999",
month = "8",
day = "3",
doi = "10.1021/bi9904609",
language = "English (US)",
volume = "38",
pages = "10024--10031",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "31",

}

Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies. / Shim, Jae Hoon; Benkovic, Stephen.

In: Biochemistry, Vol. 38, No. 31, 03.08.1999, p. 10024-10031.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies

AU - Shim, Jae Hoon

AU - Benkovic, Stephen

PY - 1999/8/3

Y1 - 1999/8/3

N2 - Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E., Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281,485-499], permitting a more exact formulation of the probable catalytic mechanism.

AB - Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E., Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281,485-499], permitting a more exact formulation of the probable catalytic mechanism.

UR - http://www.scopus.com/inward/record.url?scp=0001563512&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0001563512&partnerID=8YFLogxK

U2 - 10.1021/bi9904609

DO - 10.1021/bi9904609

M3 - Article

C2 - 10433709

AN - SCOPUS:0001563512

VL - 38

SP - 10024

EP - 10031

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 31

ER -