J. Biol. Chem., Vol. 266, Issue 23, 15021-15027, Aug, 1991

Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites

D Fabbro and GA Grabowski
Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029-6574.

Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino- 5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl-glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Locatelli-Hoops, N. Remmel, R. Klingenstein, B. Breiden, M. Rossocha, M. Schoeniger, C. Koenigs, W. Saenger, and K. Sandhoff Saposin A Mobilizes Lipids from Low Cholesterol and High Bis(monoacylglycerol)phosphate-containing Membranes: PATIENT VARIANT SAPOSIN A LACKS LIPID EXTRACTION CAPACITY J. Biol. Chem., October 27, 2006; 281(43): 32451 - 32460. [Abstract] [Full Text] [PDF]

				G. Wilkening, T. Linke, and K. Sandhoff Lysosomal Degradation on Vesicular Membrane Surfaces. ENHANCED GLUCOSYLCERAMIDE DEGRADATION BY LYSOSOMAL ANIONIC LIPIDS AND ACTIVATORS J. Biol. Chem., November 13, 1998; 273(46): 30271 - 30278. [Abstract] [Full Text] [PDF]

				A. M. Vaccaro, M. Tatti, F. Ciaffoni, R. Salvioli, A. Barca, and C. Scerch Effect of Saposins A and C on the Enzymatic Hydrolysis of Liposomal Glucosylceramide J. Biol. Chem., July 4, 1997; 272(27): 16862 - 16867. [Abstract] [Full Text] [PDF]

				X. Qi, W. Qin, Y. Sun, K. Kondoh, and G. A. Grabowski Functional Organization of Saposin C J. Biol. Chem., March 22, 1996; 271(12): 6874 - 6880. [Abstract] [Full Text] [PDF]