The use of nucleotide phosphorothioate diastereomers to define the structure of metal‐nucleotide bound to GTP‐AMP and ATP‐AMP phosphotransferases from beef‐heart mitochondria

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Abstract
The diastereomers of adenosine 5''-O-[1-thio]triphosphate (ATP[.alpha.S]) and adenosine 5''-O-[2-thio]triphosphate (ATP[.beta.S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5''-O-[thio]triphosphate (GTP[.alpha.S]) and guanosine 5''-O-[2-thio]triphosphate (GTP[.beta.S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5''-O-[1-thio]diphosphate(GDP-[.alpha.S]) were used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AKy). In all cases, coordination of Mg2+ to the .beta.-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP[.beta.S] or GTP[.beta.S], respectively, on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP[.alpha.S], or for GDP[.alpha.S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the .alpha.-phosphate group. The observed stereospecificity for the .alpha.-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the .alpha.-phosphate group with the enzyme.

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