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Description:
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As an approach to understanding the enzymological details of sterol methylation catalysis in ergosterol biosynthesis , a kinetic analysis as well as an active site mapping study of the active center of the sterol methyl transferase (SMT ) was pursued using a recombinant SMT from Saccharomyces cerevisiae . The following experiments were performed :
i . Using initial velocity conditions and equilibrium dialysis to establish the kinetic constants , Km , Vmax , and K <i , respectively , the kinetic behavior of the native SMT and a set of site -specific mutants corresponding to either the sterol or AdoMet binding site was established ,
ii . Using native and mutant SMTs that exhibit differences in the complement of Ci /C2 -activities the modulation of SMT activity by effectors such as ATP and ergosterol was determined ,
iii . Photoaffinity labeling of the active site using [^Us -methyl] AdoMet and sitespecific mutagenesis experiments were designed which successfully led to the identification of the AdoMet binding site on the SMT .
The results of these studies and that of other studies from this laboratory involving the active site characterization of the sterol binding domain , are interpreted to imply that the SMT contains an active center in which there is a sterol and AdoMet binding motif spatially disposed to allow for a random bi bi kinetic mechanism in Cmethylation of a sterol acceptor molecule . A second distinct binding center on the SMT is considered for modulators . |