The charge relay system in chymotrypsin and chymotrypsinogen

Abstract

The two buried carboxyls (Asp-102 and Asp-194) in both chymotrypsin and chymotrypsinogen are ionized at pH values greater than 4.2 and may be ionized even as low as pH 3. This was demonstrated by coupling most of the surface carboxyis of the proteins by a carbodi-imide with glycinamide or semicarbazide to diminish the groups ionizing at low pH and then titrating the proton uptake on denaturation by sodium dodecyl sulphate between pH 3.0 and 4.6. At pH values greater than 4.2 all unblocked carboxyls are ionized. The proton uptake during the conformational change on denaturation was determined by a stopped-flow procedure and found to be about 2H +/mol between pH 3.0 and 3.6. The rate constant for the uptake of protons is the same as that for the exposure of tryptophan and lies in the tens of millisecond region. The buried negative charge at the active site appears to be mainly on Asp-102 rather than on His-57, the p K a of which must be raised by the buried charge. This enhances its efficacy as a base catalyst in the “charge relay system”. The presence of an intact charge relay system in the inactive zymogen illustrates the importance of stereochemical fit between enzyme and substrate. Enzyme catalysis could hardly be mediated by a catalyst which is uniquely reactive in the absence of correct enzyme-substrate orientation as this would be inconsistent with its specificity.