Reassessment of acarbose as a transition state analogue inhibitor of cyclodextrin glycosyltransferase.

The binding of several different active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase. Michaelis-Menten parameters for the substrates alpha-maltotriosyl fluoride (alphaG3F) and alpha-glucosyl fluoride (alphaGF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an ion-selective fluoride electrode. In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants. This indicates that acarbose, as an inhibitor, is mimicking aspects of both the ground state and the transition state. A better correlation is observed for alphaGF (r = 0.98) than alphaG3F (r = 0.90), which can be explained in terms of the modes of binding of these substrates and acarbose. Re-refinement of the previously determined crystal structure of wild-type CGTase complexed with acarbose [Strokopytov, B., Penninga, D., Rozeboom, H. J., Kalk, K. H., Dijhuizen, L., and Dijkstra, B. W. (1995) Biochemistry 34, 2234-2240] reveals a binding mode consistent with the transition state analogue character of this inhibitor.