Abstract
Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.
Highlights
3.2.1.45) cleaves the glycosidic bonds of glucosyl cer- syl-1-0-@-D-glucoside:glucohydrolase,EC 3.2.1.45) cleaves amide and syntheti@ c -glucosidesC. onduritol B epoxide the @-glycosidiclinkage of glucosylceramide (GC1) (1, 2) as (CBE) and its brominated derivative are mechanism- well as synthetic @-glucosides whichare water soluble (3-5)
Kinetic studacid @-glucosidaseIgG were developed to determine ies of the purified normal human acid @-glucosidasehave the molar concentrations of functional acid B-glucosi- indicated the presence of at least three domains within its dase catalytic sites inpure placental enzyme preparations from normal sources; kcatvalues were calculated from V, = [ E t ] k tusing glucosyl ceramide substrateswith dodecanoyl (2135 f 45 min-l) and hexanoyl (3200 f 410 min-l) fatty acid acyl chains and 4-alkyl-umbelliferyl @-glucosidesubstrates with methyl (2235 f 197min-’), heptyl(l972 f 152min-’), nonyl (2220 f 247 min-l), and undecyl (773 k 44 min-l) alkyl chains
The respectivekoatvalues foracid active site which participate in substratebinding and hydrolysis: 1)the catalytic sitewhich recognizes @-glucosimdeoieties of substrates and may participate in a proton transfer for activation of substrate or for cleavage of the oxirane ring of conduritol B epoxide (CBE) for covalent binding (9); 2) an aglycon-binding site which has specificity for acyl moieties of substrates and inhibitors (9); and 3) a third domain which interacts with cationic sphingosyl derivatives as well as neg
Summary
Based inhibitors which bind covalently to the catalytic For optimal hydrolysis ofGC or synthetic substrates, this site of acid @-glucosidase.Procedures using bromine- membrane-associated enzyme requires detergents, negatively tritiated CBE and monospecific anti-human placental charged lipids, and/or a “co-glucosidase” (3-9). The respectivekoatvalues foracid active site which participate in substratebinding and hydrolysis: 1)the catalytic sitewhich recognizes @-glucosimdeoieties of substrates and may participate in a proton transfer for activation of substrate or for cleavage of the oxirane ring of conduritol B epoxide (CBE) for covalent binding (9); 2) an aglycon-binding site which has specificity for acyl moieties of substrates and inhibitors (9); and 3) a third domain which interacts with cationic sphingosyl derivatives as well as neg-. The kt the “allosteric” site described by Erickson and Radin (10).An values of the mutant splenic acid @-glucosidasefrom additional domain on theenzyme may berequired for binding two Type1Ashkenazi Jewish Gaucherdisease (AJGD) of a “co-glucosidase”(6, 7), a naturally occurring glycoprotein patients were about1.5-3-fold decreased and hadK,,, effector
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