A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites

摘要: Abstract Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms its ability to hydrolyse water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 CMC3, 60.7 microM. We designate aggregates formed at these CMCs as primary, secondary tertiary micelles respectively. From results kinetic studies performed various (0.012-248 microM), found monomers (less than 3 microM) decreased Km (for MUGlc) from 11 4.6 mM, lowered Vmax. 2-fold. However, were required for expression high activities. Glucocerebrosidase prepared patient non-neuronopathic type 1 Gaucher's disease exhibited very low (2.8 mM) even absence exogenous lipid, had no effect on mutant enzyme's or markedly increased 60% corresponding value. contrast, neuronopathic 2 case, although 9.2 1.7 never reached more 5% enzyme, sulphatide. addition, enhanced rate inactivation all preparations by chymotrypsin. Collectively, indicate presence two sulphatide-binding sites glucocerebrosidase: one enhances binding, another catalysis.