HUMAN ACID BETA-GLUCOSIDASE: FUNCTIONAL AND STRUCTURAL STUDIES OF THE NORMAL AND GAUCHER DISEASE ENZYMES.
Item
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Title
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HUMAN ACID BETA-GLUCOSIDASE: FUNCTIONAL AND STRUCTURAL STUDIES OF THE NORMAL AND GAUCHER DISEASE ENZYMES.
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Identifier
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AAI8713781
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identifier
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8713781
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Creator
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OSIECKI-NEWMAN, KAREN MARIE.
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Contributor
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Gregory A. Grabowskl
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Comparative enzymatic studies of the human acid (beta)-glucosidase ((beta)-Glc; N-acyl-sphingosyl-1-O-(beta)-glucoside:glucohydrolase, E.C.3.2.1.45) from normal placentae and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were conducted. Novel affinity chromatographic techniques were developed to obtain homogeneous normal acid (beta)-Glc and highly purified AJGD enzyme. Affinity labeling of the pure normal (beta)-Glc with the covalent, active site-directed inhibitor, conduritol B epoxide (CBE), demonstrated the presence of a single active site per subunit and permitted the identification of the active site nucleophile necessary for catalysis, Asp('443). Studies of active site function determined that the AJGD enzyme had: (1) a single active site per subunit; (2) nearly normal k(,cat) and k(,max) values for several substrates and CBE, respectively, as well as normal relative V(,max) values for glucosyl ceramides with increasing (C(,1) to C(,18)) fatty acid acyl chain lengths; (3) five to seven-fold increased K(,i)app values for particular C1 and C5 amino substituted glucosides and alkyl-glucosides (C(,0) to C(,14)) derivatives, but normal K(,i)app values for the most potent glycon inhibitors, nojirimycin and castanospermine; and (4) normal K(,m)app values for all substrates. These results were consistent with the active site of the normal enzyme being composed of three domains: a glycon binding domain which recognizes the glycon moieties of substrates and inhibitors and contains Asp('443) and a critical amino acid with pK(,a) = 6.7; an aglycon binding domain which interacts with acyl moieties and modulates V(,max); and a "third" domain which binds alkyl moieties and modulates the affinity of substrates and inhibitors. These studies also indicate that the mutation in the AJGD variant results in a unique localized effect on (beta)-Glc active site function.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
