Abstract
Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid beta-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Comparative studies were conducted with enzymes expressed from natural (spleen and fibroblasts) alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. Mutant cDNAs containing Thr43 to Lys43 (beta-GlcThr43----Lys) and Asp358 to Glu358 (beta-GlcAsp358----Glu) substitutions and two cDNAs containing Ashkenazi Jewish Gaucher disease Type 1 mutations, Arg120 to Gln120 (beta-GlcArg120----Gln) and Asn370 to Ser370 (beta-GlcAsn370----Ser) were expressed and the gene products characterized by enzymatic, immunologic, and inhibitor studies. Genotypes at the acid beta-glucosidase locus in selected Gaucher disease Type 1 patients were determined by allele-specific oligonucleotide hybridization of amplified genomic DNA. Compared with normal, recombinant or natural enzymes expressed from beta-GlcAsn370----Ser alleles had about 2-5-fold decreased specific activity based on CRIM (cross-reacting immunologic material). The beta-GlcArg120----Gln cDNA expressed catalytically inactive CRIM in Sf9; consistent with the 9-fold decreased CRIM-specific activity of the natural enzyme from a beta-GlcArg120----Gln/beta-GlcAsn370----Ser genetic compound. The beta-GlcAsp358----Glu cDNA expressed catalytically inactive CRIM in Sf9 cells. The presence of natural or recombinant enzyme expressed from beta-GlcAsn370----Ser alleles was sufficient to confer 3-5-fold increased IC50 values for deoxynojirimycin, glucosylsphingosine, and N-alkyl-glucosylamine derivatives. Progress curves for inhibition by the slow-tight binding N-alkyl-glucosylamines indicated that the beta-Glc-Asn370----Ser mutation did not alter a conformational change induced by these reaction intermediate analogues. These results provide evidence that the beta-GlcArg120----Gln and beta-GlcAsn370----Ser mutations found in Gaucher disease Type 1 patient genomes are the molecular bases of the enzymatic dysfunction. In addition, the region including Arg120 and that encompassing Asp358 and Asn370 contain residues critical to active site formation or participation in the catalytic mechanism.
Highlights
From the *Division of Medical and Molecular Genetics and the Departments School of Medicine, New York, New York 10029 of llMedicine, A
Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid /3-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles
Comparative studies were conducted with enzymes expressed from natural alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system
Summary
Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid /3-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Additional residues were shown to participate in catalysis and/or substrate binding, based on kinetic data, but they have not been identified (e.g. Basu et al, 1985; Osiecki-Newman et al, 1987, 1988) The nature of these residues within the active site and effector sites is important for understanding the basis of the acid P-glucosidase defects in the variants of Gaucher disease. We used site-directed mutagenesis and expression of mutagenized cDNAs encoding acid /3-glucosidases to investigate the functional defects which result from two point mutations identified in Gaucher disease Type 1 in the Ashkenazi Jewish population. The results of selected kinetic and immunoblotting analyses provide insight into the nature of catalytically necessary residues for normal enzymatic function These findings account for the presence of catalytic activity in tissues and the defective function of the residual acid /3-glucosidase in Ashkenazi Jewish Gaucher disease Type
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