Protection of hexaprenyl-diphosphate synthase of Micrococcus luteus B-P 26 against inactivation by sulphydryl reagents and arginine-specific reagents.

Hexaprenyl-diphosphate synthase from Micrococcus luteus B-P 26 has been shown to comprise two essential components, designated as components A and B. Treatment of the synthase with sulphydryl reagents (N-ethylmaleimide, iodoacetamide or p-chloromercuribenzoate) or arginine-specific reagents (2,3-butanedione, 1,2-cyclohexanedione or phenylglyoxal) resulted in a rapid loss of the component B activity. In contrast, component A was resistant to treatment with such reagents, retaining the initial activity almost completely. Farnesyl diphosphate, isopentenyl diphosphate, farnesyl monophosphate and inorganic pyrophosphate protected the synthase against the inactivation by N-ethylmaleimide, farnesyl diphosphate being the most effective. The presence of Mg2+ was essential for the protection by isopentenyl diphosphate and inorganic pyrophosphate. For protection of the synthase activity against the inactivation by 2,3-butanedione, the presence of farnesyl diphosphate, isopentenyl diphosphate and Mg2+ was more effective than that of the individual substrates and Mg2+. Inorganic pyrophosphate provided substantial protection. In the absence of component A, the component B activity was not protected by any substrates or its analogue. These results suggest that the catalytic site of the synthase is formed by cooperative interaction between components A and B, and that cysteine and arginine residues on component B play important roles in the synthase activity.