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Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid ß-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., K(is) < < K(ii)) and had low apparent affinity for the enzyme (K(is)app = 20-500 mmol/l). ß-Glucose- 1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-ß-glucoside hydrolysis in an uncompetitive pattern (K(i)app = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-ß-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5- imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with K(is)app(ß-glucose)/K(is)app ≅ 10^3-10^5; an amine at C-2 did not alter K(is)app compared to ß-glucose. The variation of K(is)app with pH for the 5-imino- and 1- deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH(2)) of the diprotic (V(max)app and V(max)app/K(m)app) normal enzyme (pK(1) = 4.7; pK(2) = 6.7) with pH-independent K(is)app values of 2.9-9.0 µmol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that K(is)app(AJGD)/K(is)app(normal) = 4-17 for dNM and 1- deoxy-1-amino-ß-glucose; this ratio was ≅ 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid ß-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of ß-glucose and a residue with pK(a) = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid ß-glucosidase.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(l l-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid ß-glucosidase (ß-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal ß-Glc/ml of settled gel, respectively. The purified normal enzyme (14- 18% yield) had a specific activity of 1.6 X 10^6 nmol/h/mg protein and was homogeneous as evidenced by a single protein species of M(r) = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the ß-Glc cDNA. Amino acid composition analyses of ß-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11 %. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

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