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 investi gated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles i n governing the product specificity of cyclodextrin glycosyltransferase. Mi chaelis-Menten parameters for the substrates alpha-maltotriosyl fluoride (a lpha G3F) and alpha-glucosyl fluoride (alpha GF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an i on-selective fluoride electrode. In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both k(cat)/K-m and K-m 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 alpha GF (r = 0.98) than alpha G3F (r = 0.90), which can be explained in terms of the modes of binding of these su bstrates and acarbose. Re-refinement of the previously determined crystal s tructure of wild-type CGTase complexed with acarbose [Strokopytov, B., Penn inga, D., Rozeboom, H. J., Kalk, K. H., Dijhuizen, L., and Dijkstra, B. W. (1995) Biochemisrry 34, 2234-2240] reveals a binding mode consistent with t he transition state analogue character of this inhibitor.