Some aspects of the mechanism of complexation of red kidney bean .alpha.-amylase inhibitor and .alpha.-amylase

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Abstract
Bovine pancreatic .alpha.-amylase binds 1 mol of acarbose (a carbohydrate .alpha.-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean .alpha.-amylase inhibitor-bovine pancreatic .alpha.-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl .alpha.-D-maltoside to the final complex of red kidney bean .alpha.-amylase inhibitor and bovine pancreatic .alpha.-amylase has a .beta.Ks (Ks'') value that is 3.4-fold greater than the Ks (16 mM) of .alpha.-amylase for p-nitrophenyl .alpha.-D-maltoside alone. The initial complex of .alpha.-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as .alpha.-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of .alpha.-amylase, caused dissociation of the red kidney bean .alpha.-amylase inhibitor-.alpha.-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) .alpha.-amylase inhibitor and porcine pancreatic .alpha.-amylase proceeds through 2 steps. The first step has a Keq of 3.1 .times. 10-5 M. The 2nd step (unimolecular; 1st order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30.degree. C. .alpha.-Amylase inhibitor combines with .alpha.-amylase, in the presence of p-nitrophenyl .alpha.-D-maltoside, noncompetitively. .alpha.-Amylase is inactivated by the .alpha.-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow tight-binding inhibitors that proceed through 2 steps.