Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group.
The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.
Mullins
L S
LS
Department of Chemistry, Texas A&M University, College Station 77843.
Lusty
C J
CJ
Raushel
F M
FM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D000251:Adenosine Triphosphatases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D003545:Cysteine