Várallyay, E., Lengyel, Z., Gráf, L. & Szilágyi, L.

The role of disulfide bond C191-C220 in trypsin and chymotrypsin.

Biochem Biophys Res Commun 230, 592-6.

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/KM of 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 pKa in 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.

Kaslik, Gy., Kardos, J., Szabó, E., Szilágyi, L., Závodszky, P., Westler, M., Markley, J. L., Gráf, L.

Effect of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

Biochemistry 36, 5455-5464.