ASSESSMENT OF THE ROLE OF AN OMEGA-LOOP OF CHOLESTEROL OXIDASE - A TRUNCATED LOOP MUTANT HAS ALTERED SUBSTRATE-SPECIFICITY

The function of an active site loop (70-90) of cholesterol oxidase has been ascertained by deleting five contiguous residues (79-83) from th e tip of the loop. From the crystal structure of the wild-type enzyme, it appears that this truncation will not significantly perturb the st ructure of the rest of the enzyme. The UV/vis and CD spectra of the mu tant confirm that the enzyme is properly folded with FAD bound. The mu tant enzyme still transfers H-2 from the 4 beta-carbon of the intermed iate, cholest-5-en-3-one, to the 6 beta-carbon of the product, cholest -4-en-3-one, during isomerization. The k(cat)/K-m of the mutant is inc reased 6-fold with dehydroepiandrosterone as substrate. Thus, the enzy me is still catalytically active after deletion of the five loop-tip r esidues. With micellar cholesterol, the k(cat)/K-m of the mutant is de creased 170-fold relative to wild type. This suggests that the tip of the loop is necessary for packing with the ''tail'' of cholesterol and is responsible for substrate specificity at C-17. Increased release o f intermediate cholest-5-en-3-one in the mutant-catalyzed reaction is not observed. Truncation of the loop, therefore, does not affect the g rip of the enzyme on the intermediate. With lipid vesicle substrates ( egg phosphatidylcholine/cholesterol, 1:1), the initial velocity of the mutant is reduced 3000-fold. The binding affinity for the vesicles, h owever, is only reduced 2-fold. Consequently, the loop is not the prim ary determinant of binding affinity for vesicles. It is concluded that the loop is important for movement of cholesterol from the lipid bila yer. The tip residues form a hydrophobic pathway between lipid membran e and active site to facilitate movement of substrate and product in t o and out of the active site.