Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase.
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Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococcus faecalis), unadenylylated glutamine synthetase (Escherichia coli), and carbamoyl-phosphate synthetase (E. coli). 31P NMR was used to determine that carbamate kinase uses the B isomer of Ado-5-(2-thioPPP) in the presence of Mg2+. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd2+ suggesting that the metal ion does not bind to the -phosphoryl group or that both Mg2+ and Cd2+ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5-(1-thioPP) with Mg2+ and Cd2+. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5-(2-thioPPP) at both ATP sites with Mg2+ (Raushel et al., 1978 J. Biol. Chem. 253, 6627). Current experiments show that the stereospecificity is reversed by Cd2- and that both A and B isomers are used when Zn2+ is present. With Ado-5-(1-thioPPP), the B isomer is used with Mg2+, the A isomer with Cd2+, and both isomers with Zn2+. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH3)4ATP as a substrate and thus we can only speculate that the chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co3+-ATP analogs utilized by hexokinase (E. K. Jaffe, and M. Cohn, 1978 Biochemistry 17, 652). If the sulfur of the -phosphoryl of Ado-5-(2-thioPPP) binds to the metal ion with carbamate kinase, then the chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Glutamine synthetase reacts with the B isomer of both Ado-5-(2-thioPPP) and Ado-5-(1-thioPPP) in the presence of Mg2+. When Co2+ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH3)4ATP is not a substrate for glutamine synthetase. Glutamine synthetase is therefore different from the two previously mentioned enzymes in that it used the opposite A ring configuration for the metal-ATP chelate. 1980.
