Phosphorothioate analogues of adenosine triphosphate as substrates of the mevalonate kinase reaction

Abstract

Adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) and the diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP[alpha S]) and adenosine 5'-O-(2-thiotriphosphate) (ATP[beta S]) could act as substrates for mevalonate kinase (ATP: mevalonate 5-phosphotransferase, EC 2.7.1.36) in the presence of different activating divalent metal cations. In the presence of Mg2+ the Rp diastereomers of both ATP[alpha S] and ATP[beta S] were the preferred substrates, whereas in the presence of Cd2+ the Sp diastereomers were the more active. Calculation of Vmax/Km values confirmed reversal of the Rp/Sp ratios for ATP[beta S] with Mg2+ [16] and Cd2+ [2], providing evidence that the metal is coordinated to the beta-phosphate at the active site. However, the corresponding ratios were very similar for ATP[alpha S], 0.4 and 0.2 for Mg2+ and Cd2+, respectively, so that it is unlikely that the metal ion was coordinated to the alpha-phosphate, in the active complex. The Vmax for CdATP [gamma S] was some 90-fold less than that for MgATP[gamma S], suggesting that Cd2+ is bound to S from the gamma-P and that the breaking of the Cd-S bond is the rate determining step of the reaction. It was concluded that the chelate structure of metal ATP substrate in the mevalonate kinase reaction is the lambda,beta,gamma-bidentate MgATP chelate. The probable product of the reaction is delta,alpha,beta-bidentate MgADP.