RESIDUE ACCESSIBILITY, HYDROGEN-BONDING, AND MOLECULAR RECOGNITION - METAL-CHELATE PROBING OF ACTIVE-SITE HISTIDINES IN CHYMOTRYPSINS

Subspecies defining the maturation pathway of bovine chymotrypsinogen to alpha-chymotrypsin have been separated in a single chromatographic run by affinity to iminodiacetic acid-Cu(II) [IDA-Cu(II)] immobilized onto Novarose. A major highlight of the elution pattern is that, as ma turation proceeds, these subspecies exhibit a correlated increase in a ffinity toward IDA-Cu(II). This behavior is analyzed by a combination of physicochemical and molecular modeling techniques to assess the con tribution of the two histidines present in chymotrypsins, at positions 40 and 57 on the protein surface. Catalytic His-57 features adequate surface accessibility to serve as a ligand to IDA-Cu(II), but its part icipation is clearly ruled out by specific chemical modification. In c ontrast, His-40, whose side chain is buried in the crystal structures of both zymogen and mature enzyme, surprisingly proves the most plausi ble candidate as an electron donor to IDA-Cu(II). This apparent confli ct between histidine accessibility and their implication in IDA-Cu(II) recognition has been rationalized on the basis of their flexibility a nd/or hydrogen-bonding status, with the following outcome. First, hist idine constitutes a useful reporter group for subtle protein conformat ional fluctuations. Second, static accessibility computation alone pro vides no unequivocal guideline as to whether a protein residue can ser ve as a ligand. Third, this study is the first to document the occurre nce of a screening effect due to hydrogen bonding of an otherwise ''ac cessible'' histidine. A significant corollary to this finding would be that the catalytic histidine is rigidly entrapped in a remarkably str ong hydrogen-bonding network, a situation that may pertain to mechanis tic aspects of catalysis.