CALCULATION OF SUBSITE AFFINITIES OF HUMAN SMALL-INTESTINAL GLUCOAMYLASE-MALTASE

Several years ago, Hiromi at al. (1973) Biochim. Biophys. Acta 302, 362-375 proposed a theory for the action patterns of glucoamylases, based on data from steady-state kinetics. The Michaelis-Menten constants (K-m) and the turnover number for maltooligosaccharides were used to evaluate the subsite affinities. We have now used this method for evaluating the subsite affinities for glucoamylase(EC 3.2.1.3)-maltase (EC 3.2.1.20) from human intestinal mucosa. For calculation of the subsite affinities, A(1) and A(2), and the intrinsic rate constant k(int), we use a modified algorithm and a computer program for nonlinear least square fitting. Considerable substrate inhibition was shown by maltotriose, minor inhibition by maltotetraose, and no inhibition by maltose and the other maltooligosaccharides. This indicates a more complex kinetic behaviour of the enzyme with respect to maltotriose. Evaluation of the subsites reveals that A(2) is the main binding site (18.1 kJ/mol), whereas the other affinities, with the exception of A(1), are lower than 2.5 kJ/mol.