THE ROLE OF DISULFIDE BOND C191-C220 IN TRYPSIN AND CHYMOTRYPSIN

Serine proteases of the chymotrypsin family contain three conserved di sulfide bonds: C42-C58, C168-C182, and C191-C220. C191-C220 connects t he loops around the substrate binding pocket. Using site directed muta genesis, cysteines of this disulfide bridge were replaced by alanines in trypsin, in chymotrypsin, and in Tr-->CH-[S1+L1+L2+Y172W], a mutant trypsin with high chymotrypsin like activity. The functional role of this ''active site'' disulfide was assessed by comparing the catalytic properties of wild-type and mutant enzymes. Its removal from all thre e proteases caused a decrease in k(cat)/K-M of two to three orders of magnitude, mainly as a consequence of a dramatic increase in K-M. The pH dependence of the activity also changed: the rather wide pH optimum , characteristic of the wild-type enzymes (especially trypsin), narrow ed since the pK(a) in the alkaline region shifted downwards. Results s how that C191-C220 is necessary for the high activity of both trypsin and chymotrypsin. By contrast, elimination of this disulfide bridge gr eatly decreased the specificity of trypsin and of Tr-->Ch-[S1+L1+L2+Y1 72W], but had no significant change on that of chymotrypsin. (C) 1997 Academic Press