INFRARED SPECTROSCOPIC STUDIES OF CALCIUM-BINDING TO INHIBITED BETA-TRYPSINS

Fourier transform infrared spectroscopy was used to examine the effect of calcium binding on the secondary structure of two inhibited bovine beta-trypsins. Neither the diisopropyl fluorophosphate- nor benzamidi ne-inhibited forms showed detectable secondary structure perturbation upon calcium binding at pD 6.9 and 5.0, respectively. Considered in li ght of the recent assignment of an amide I' band to the autolysis loop of bovine beta-trypsin, these results contradict the generally held h ypothesis that calcium slows trypsin autolysis by induction of a confo rmational change at this site and support the recent contention that t he mechanism of action has a specific electrostatic origin. In additio n, the appearance of a band at 1699 cm(-1) in the benzamidine-inhibite d form can be interpreted as resulting from the NC-N stretching vibrat ions of the amidinium moiety, which the observed crystal structure ind icates is hydrogen-bonded to the carboxyl group of active-site Asp-189 .