Abstract
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 Mg 2+. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd 2+ suggesting that the metal ion does not bind to the β-phosphoryl group or that both Mg 2+ and Cd 2+ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5′-(1-thioPP) with Mg 2+ and Cd 2+. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5′-(2-thioPPP) at both ATP sites with Mg 2+ ( Raushel et al., 1978 J. Biol. Chem. 253, 6627). Current experiments show that the stereospecificity is reversed by Cd 2− and that both A and B isomers are used when Zn 2+ is present. With Ado-5′-(1-thioPPP), the B isomer is used with Mg 2+, the A isomer with Cd 2+, and both isomers with Zn 2+. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH 3) 4ATP as a substrate and thus we can only speculate that the Δ chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co 3+-ATP analogs utilized by hexokinase ( E. K. Jaffe, and M. Cohn, 1978 Biochemistry 17, 652). If the sulfur of the β-phosphoryl of Ado-5′-(2-thioPPP) binds to the metal ion with carbamate kinase, then the Δ 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 Mg 2+. When Co 2+ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH 3) 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.
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