Substitution of Glu841 by lysine in the carbamate domain of carbamyl phosphate synthetase alters the catalytic properties of the glutaminase subunit.
In previous studies a Glu841-->Lys replacement in the carbamate phosphorylating domain located in the COOH half of the synthetase subunit of Escherichia coli carbamyl phosphate synthetase was shown to reduce overall synthesis of carbamyl phosphate by 4 orders of magnitude with either glutamine or NH3 as nitrogen donor (Guillou et al., 1992). In the present study, the mutant enzyme has been further analyzed for its glutamine hydrolytic activity. The glutaminase activity of the mutant enzyme has the following properties. (1) In the absence of other substrates the turnover number is only marginally different from that of the wild-type complex. (2) The Km for glutamine is 60 times higher than in wild-type complex and three times higher than in the separated glutaminase subunit. (3) In the present study wild-type carbamyl phosphate synthetase has been shown to catalyze glutamine hydrolysis by a mechanism involving an enzyme-bound acyl ester intermediate (gamma-glutamyl thioester). This intermediate is formed and is hydrolyzed with rates consistent with overall glutamine hydrolysis. At physiological concentrations of glutamine (1.2 mM), the steady-state concentration of gamma-glutamyl thioester is 0.3 mol/mol of wild-type enzyme. Under the same conditions, only 0.02 mol of thioester is measured in the mutant enzyme. Maximal accumulation of this covalent intermediate by the mutant enzyme required 10 times higher concentrations of free glutamine. (4) The rate of reaction with 2-amino-4-oxo-5-chloropentanoate, a glutamine analog known to specifically alkylate an active site cysteine residue, is 2 orders of magnitude slower in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
Lusty
C J
CJ
Department of Molecular Genetics, Public Health Research Institute, New York, New York 10016.
Liao
M
M
D000595:Amino Acid Sequence; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D005972:Glutaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008433:Mathematics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D005971:Glutamates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018698:Glutamic Acid; D005972:Glutaminase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008239:Lysine