Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

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The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54766-9 ◽

1991 ◽

Vol 266 (31) ◽

pp. 20709-20713

Author(s):

L.J. Donald ◽

B.R. Crane ◽

D.H. Anderson ◽

H.W. Duckworth

Keyword(s):

Escherichia Coli ◽

Chemical Modification ◽

Citrate Synthase ◽

Site Directed Mutagenesis ◽

19F Nmr ◽

Directed Mutagenesis ◽

Allosteric Inhibition

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Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

Biochimie ◽

10.1016/j.biochi.2007.06.011 ◽

2007 ◽

Vol 89 (12) ◽

pp. 1498-1508 ◽

Cited By ~ 18

Author(s):

Muriel Crouvoisier ◽

Geneviève Auger ◽

Didier Blanot ◽

Dominique Mengin-Lecreulx

Keyword(s):

Amino Acid ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

Biochemical Journal ◽

10.1042/bj3010477 ◽

1994 ◽

Vol 301 (2) ◽

pp. 477-483 ◽

Cited By ~ 9

Author(s):

J M Wilkin ◽

A Dubus ◽

B Joris ◽

J M Frère

Keyword(s):

Amino Acids ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Peptide Substrate ◽

Binding Properties ◽

Beta Lactamases ◽

Penicillin Binding Proteins ◽

Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

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