Disulfide-linked propeptides stabilize the structure of zymogen and maturepancreatic serine proteases

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with p ropeptides that remain attached to the active enzyme via a disulfide bridge . It is likely, although not proven, that these propeptides are functionall y important in the active enzymes, as well as in the zymogens. A mutant chy motrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., ct al. (1996 ) FEES Lett. 379, 139-142]. In this paper, we investigate the role of the d isulfide-linked propeptide in the conformational stability of chymotrypsin( ogen). We compare the stabilities of the wild-type and mutant proteins (lac king propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) an d active (chymotrypsin) forms. The mutants exhibited a substantially increa sed sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experime nts, we determined that covalently linked propeptide provides about 24 kJ/m ol of free energy of extra stabilization (Delta Delta G). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin . This may also explain (besides keeping the zymogen inactive) the evolutio nary conservation of the propeptide-enzyme interactions. Tryptophan fluores cence, circular dichroism, microcalorimetric, and activity measurements sug gest that the propeptide of chymotrypsin restricts the relative mobility be tween the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appear s to be accomplished via alternative interdomain contacts.