The effects of chemical modification on the refolding transition of alpha-chymotrypsin.

The role of several active site residues of alpha-chymotrypsin in the prototypical refolding transition between active and inactive forms of this enzyme is examined using chemical modification. Oxidation of Met-192 to the sulfoxide results in a derivative which remains entirely in an active state from pH 6 to 9. The derivative becomes inactive only at high pH with pKa = 10.3, delta H0 = 9.5 kcal and delta S0 = -15 eu., indicating the sulfoxide group supplies about 2.1 kcal of active state stabilization relative to the unoxidized methionine side chain. The refolding transition of N-methyl-His-57-alpha-chymotrypsin, in which a nitrogen of the "charge relay" histidine is methylated, displays one ionization process with an apparent pKa of 9.45. The absence of an additional ionization process with a pKa near 7 provides evidence that one of the ionizations in the six state mechanism which describes this transition in alpha-chymotrypsin is linked to the charge relay system. We also demonstrate, using alpha-chymotrypsin, Met-192-sulfoxide-alpha-chymotrypsin and N-methyl-His-57-alpha-chymotrypsin, that the 230 nm circular dichroism band is a quantitative probe of the active-inactive equilibrium, although the chromophore or chromophores responsible for this and another very large negative band at 202 nm have not been identified. Circular dichroism was used to observe the active-inactive equilibrium in methan sulfonyl-alpha-chymotrypsin and phenylmethane sulfonyl-alpha-chymotrypsin. The enhanced stability of the active state of these derivatives relative to alpha-chymotrypsin can be rationalized in terms of steric effects in the substrate side chain binding site.