Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447.
Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.
Yin
Ye
Y
Department of Chemistry, State University of New York, Stony Brook, New York 11794-3400, USA.
Liu
Pingsheng
P
Anderson
Richard G W
RG
Sampson
Nicole S
NS
D001665:Binding Sites; D021941:Caveolae / Q000378:metabolism / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D008051:Lipid Bilayers; D018698:Glutamic Acid; D006639:Histidine; D002789:Cholesterol Oxidase