We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.

A burst of release of one equivalent of trinitrophenolate observed upon inactivation of human pancreatic ␣-amylase proves the required 1:1 stoichiometry. These are the first mechanism-based inhibitors of this class to be described, and the first mechanism-based inhibitors of any sort for the medically important ␣-amylase. In addition to having potential as therapeutics, compounds of this class should prove useful in subsequent structural and mechanistic studies of these enzymes.Specific inhibitors of glycosidases have proved valuable in a number of applications ranging from mechanistic studies (Legler, 1990;Sinnott, 1990) through their use to study protein glycosylation (Elbein et al., 1984), to possible therapeutic uses such as the control of blood glucose levels via control of the degradation of dietary disaccharides and starch (Truscheit et al., 1981) or control of viral infectivity through interference with normal glycosylation of viral coat proteins (Elbein, 1984;Prasad et al., 1987). A number of naturally occurring reversible glycosidase inhibitors are known such as nojirimycin, castanospermine, swainsonine, and acarbose (Legler, 1990), and these have been subjected to intensive study including the synthesis and testing of a number of analogues. Another class of inhibitors that has been less well studied is that of the covalent, irreversible type, typically affinity labels. These are generally synthetic analogues of sugars containing reactive groups such as epoxides, isothiocyanates and ␣-halocarbonyls as reviewed recently (Legler, 1990;Withers and Aebersold, 1995). Less common are the more selective mechanism-based inhibitors whose efficacy depends upon binding and subsequent enzymatic action to generate a reactive species. These include the conduritol epoxides (Legler, 1968(Legler, , 1970, the quinone methidegenerating glycosides (Halazy et al., 1990;Briggs et al., 1992), and the glycosylmethyl triazenes (Marshall et al., 1980;Sinnott and Smith, 1976). Interestingly, two naturally occurring inhibitors of this class have now been described: the hydroxymethylconduritol epoxide, cyclophellitol (Atsumi et al., 1990;Withers and Umezawa, 1991), isolated from Phellinus sp.; and the putative quinone methide-generating glycoside salicortin, isolated from Salix (Clausen et al., 1990).An additional, relatively recently described class of mechanism-based inhibitor that has proved successful is that of the 2-deoxy-2-fluoro (Withers and Aebersold, 1995;Withers et al., 1987Withers et al., , 1988Withers et al., , 1990. These function as excellent inactivators of retaining glycosidases (glycosidases that hydrolyze the glycosidic linkage with net retention of anomeric configuration) by formation of a stable glycosyl-enzyme intermediate which turns over to product only very slowly. As shown in Scheme 1 for an ␣-glucosidase, the normal mechanism of action of this class of enzyme involves the formation and hydrolysis of a glycosyl-enzyme intermediate with general acid/base catalytic assistance via transition states with subs...

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonasfimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are >60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulosebinding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the ,3-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.

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The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)