COOPERATIVE INTERACTIONS OF THE CATALYTIC NUCLEOPHILE AND THE CATALYTIC ACID IN THE INHIBITION OF BETA-GLYCOSIDASES - CALCULATIONS AND THEIR VALIDATION BY COMPARATIVE KINETIC AND STRUCTURAL STUDIES OF THE INHIBITION OF GLYCOGEN-PHOSPHORYLASE-B

The difference between the strong inhibition of retaining beta-glucosi dases by the tetrazole 1 and the weak inhibition by the triazole 3 has been explained by the protonation by the enzymic catalytic acid of N( 3) of 1, replaced by CH in 3. One also expects a contribution to the i nhibition from the charge-dipole interaction between the enzymic catal ytic nucleophile and the azole ring. The extent of this contribution w as estimated from the calculated, distance-dependent heats of formatio n of the acetate-azole complexes. The calculations were validated by c omparison of the charge-dipole interaction between phosphate and the i nhibitors 1 and 3 in the glycogen phosphorylase b (GPb)-azole-phosphat e complexes, as derived from differences in the K-i values for I and 3 , while the structural invariance of the complexes was demonstrated by X-ray analysis. The difference between the charge-dipole interactions of (dihydrogen)phosphate and 1 or 3 as derived from Delta K-i is 1.1 kcal mol(-1), while the calculated difference is 1.3 kcal mol(-1). The calculated difference for the interaction of 1 or 3 with acetate, rep resenting the catalytic nucleophile in beta-glycosidases, is 2.0 kcal mol(-1), while the differences of the binding energies as derived from the K-i values for the inhibition by 1 or 3 of different beta-glycosi dases range from 2.4 to 5.3 kcal mol(-1) The calculated difference for 1 and the imidazole 6 is 2.5 kcal mol(-1) in favour of 1, whereas the K-i-derived difference is 3.7 kcal mol(-1) in favour of 6, equal to t he calculated difference between 1 and the protonated imidazole 6. Thu s, protonation by the catalytic acid and the charge-dipole interaction with the catalytic nucleophile contribute cooperatively to the bindin g of inhibitors possessing a trigonal anomeric centre bonded to a hete roatom.