Mechanism-based Inhibition of Yeast α-Glucosidase and Human Pancreatic α-Amylase by a New Class of Inhibitors

Abstract

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...

Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

“…Conceivably, D1136 pushes the −1 O2 atom towards the intermediate position, while Q1509 holds the −1 glucose O6 atom. Similar to GH13 enzymes, the C2-O2 bond of the −1 glucose residue is polarized by interactions of the O2 atom with H1135, D1136, and R1023, which lowers the energy of the transition state (14,22,33,34). The reaction is completed by nucleophilic attack of D1025 on the glucose C1 atom, which results in a covalent intermediate with the glucosyl moiety attached to D1025 via a β-glycosidic linkage.…”

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“…Hydrogen bonding interactions at this position can contribute up to 48 kJ mol -1 to transition state stabilization 18,19 . These hydrogen bonds serve two purposes.…”

“…One is the distortion of the sugar ring towards the half chair conformation. The other is a reduction in the electronegativity of the 2-hydroxyl, which otherwise strongly disfavors formation of a positively charged transition state 18,19 . CGTase reduces this electronegativity in two ways.…”

Toward the Virtual Screening of α-Glucosidase Inhibitors with the Homology-Modeled Protein Structure

“…The calculated binding modes of the two inhibitors in the active site of α-glucosidase are compared in Figure 6. It is seen that the 5,7-dihydroxy- [1,4]naphthoquinone moiety of 1 resides in close proximity to the catalytic residues including Asp214, Glu276, His348, and Asp349, indicating that it can serve as a surrogate for the terminal glucose with anomeric center in the substrate. Four hydrogen bonds are established between the phenolic and carbonyl oxygens of 1 and the side chains of Asp214, Thr215, Ser244, and Arg312.…”

“…Of the two popular glucosidases, α-glucosidase (EC 3.2.1.20) has drawn a special interest of the pharmaceutical research community because it was shown in earlier studies that the inhibition of its catalytic activity resulted in the retardation of glucose absorption and the decrease in postprandial blood glucose level. [3][4][5] Therefore, effective α-glucosidase inhibitors may serve as chemotherapeutic agents for clinic use in the treatment of diabetes and obesity. Due to the catalytic role in digesting carbohydrate substrates, α-glucosidase has also been well appreciated as a therapeutic target for the other carbohydrate mediated diseases including cancer, 6 viral infections, 7,8 and hepatitis.…”