Abstract
Serine proteases of the chymotrypsin family contain three conserved disulfide bonds: C42-C58, C168-C182, and C191-C220. C191-C220 connects the loops around the substrate binding pocket. Using site directed mutagenesis, 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 three proteases caused a decrease in kcat/KMof two to three orders of magnitude, mainly as a consequence of a dramatic increase in KM. The pH dependence of the activity also changed: the rather wide pH optimum, characteristic of the wild-type enzymes (especially trypsin), narrowed since the pKain the alkaline region shifted downwards. Results show that C191-C220 is necessary for the high activity of both trypsin and chymotrypsin. By contrast, elimination of this disulfide bridge greatly decreased the specificity of trypsin and of Tr→Ch[S1+L1+L2+Y172W], but had no significant change on that of chymotrypsin.
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