Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase.

Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococots faecalis), unadenylylated glutamine synthetase (Escherichia co(i). and carbamoyl-phosphate synthetase (E. coli). :llP NMR ~vvas used to determine that carbamate kinase uses the B isomer of Ado-5’.(2-thioPPP) in the presence of MgY’. The stereospecificity of the reaction with carbamate kinase was not reversed by CdY+ suggesting that the metal ion does not bind to the P-phosphoryl group or that both Mg” and Cd 2+ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5’.(1-thioPP) with Mg’+ and CdZ+. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5’-(SthioPPP) at both ATP sites with Mg’+ (Raushelet nl., 19781. Biol. Chem. 253,6627). Current experiments show that the stereospecificity is reversed by Cd2* and that both A and B isomers are used when Zn’+ is present. With Ado-5’.(l-thioPPP), the B isomer is used with Mg’+. the A isomer with Cd”, and both isomers with Zn”. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH:,),ATP as a substrate and thus \ve can only speculate that the A chelate ring configuration is the chelatr structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co’“-ATP analogs utilized by hexokinase (E. I<. Jaffe, ant1 >l. Cohn, 19%Biocl/e)i?istqj 17, 652). If the sulfur of the fl-phosphoryl of Ado-5’.(2.thioPPP) binds to the metal ion with carbamate kinase, then the A chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate sgnthetase. Glutamine synthetase reacts with the B isomer of both Ado-5’.(2.thioPPP) and Ado-5’-(l-thioPPP) in the presence of MgY*. When Co” is used with this enzyme the A and B isomers of both thioATP compounds are substrates. Co(III)(NH,,),ATP 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.