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 the tip of the loop. From the crystal structure of the wild-type enzyme, it appears that this truncation will not significantly perturb the structure of the rest of the enzyme. The UV/vis and CD spectra of the mutant confirm that the enzyme is properly folded with FAD bound. The mutant enzyme still transfers 2H from the 4beta-carbon of the intermediate, cholest-5-en-3-one, to the 6beta-carbon of the product, cholest-4-en-3-one, during isomerization. The kcat/Km of the mutant is increased 6-fold with dehydroepiandrosterone as substrate. Thus, the enzyme is still catalytically active after deletion of the five loop-tip residues. With micellar cholesterol, the kcat/Km of the mutant is decreased 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 C17. Increased release of intermediate cholest-5-en-3-one in the mutant-catalyzed reaction is not observed. Truncation of the loop, therefore, does not affect the grip 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, however, is only reduced 2-fold. Consequently, the loop is not the primary determinant of binding affinity for vesicles. It is concluded that the loop is important for movement of cholesterol from the lipid bilayer. The tip residues form a hydrophobic pathway between lipid membrane and active site to facilitate movement of substrate and product in to and out of the active site.
Sampson
N S
NS
Department of Chemistry, State University of New York, Stony Brook 11794-3400, USA. nicole.sampson@sunysb.edu
Kass
I J
IJ
Ghoshroy
K B
KB
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001951:Brevibacterium / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D002789:Cholesterol Oxidase / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D008051:Lipid Bilayers; D003903:Deuterium; D002789:Cholesterol Oxidase