Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis.
The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.
Ator
M A
MA
Department of Medicinal Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406-0939.
Schmidt
S J
SJ
Adams
J L
JL
Dolle
R E
RE
D002175:Candida / Q000187:drug effects / Q000201:enzymology*; D002176:Candida albicans / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D005285:Fermentation; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D012995:Solubility; D013261:Sterols / Q000494:pharmacology; D013379:Substrate Specificity
D013261:Sterols; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase