Pig Liver Phosphomevalonate Kinase. 2. Participation of Cysteinyl and Lysyl Groups in Catalysis

Abstract

Phosphomevalonate kinase from pig liver is inactivated by 5,5'-dithiobis(2-nitrobenzoate) and pyridoxal 5'-phosphate. The substrate phosphomevalonate protects the enzyme against inactivation by these reagents. Inactivation by 5,5'-dithiobis(2-nitrobenzoate) is complete and may be reverted by 2-mercaptoethariol or dithiothreitol. Experiments carried out with partially inactivated enzyme show no change in the kcat or in the apparent Km for the Substrates, as compared with the native enzyme, indicating the existence of two populations of molecules, one intact and the other totally inactive. These results suggest that 5,5'-dithiobis(2-nitrobenzoate) reacts with the only cysteinyl residue of the enzyme and that this residue is located in or near the active site. Inhibition by pyridoxal 5'-phosphate can be reverted, either by dialysis or by the addition of lysine, but not if the partially inactivated enzyme is treated previously with NaBH4, in agreement with the formation of a Schiff base be