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 maturation proceeds, these subspecies exhibit a correlated increase in affinity toward IDA-Cu(II). This behavior is analyzed by a combination of physicochemical and molecular modeling techniques to assess the contribution 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 participation is clearly ruled out by specific chemical modification. In contrast, His-40, whose side chain is buried in the crystal structures of both zymogen and mature enzyme, surprisingly proves the most plausible candidate as an electron donor to IDA-Cu(II). This apparent conflict between histidine accessibility and their implication in IDA-Cu(II) recognition has been rationalized on the basis of their flexibility and/or hydrogen-bonding status, with the following outcome. First, histidine constitutes a useful reporter group for subtle protein conformational fluctuations. Second, static accessibility computation alone provides no unequivocal guideline as to whether a protein residue can serve as a ligand. Third, this study is the first to document the occurrence of a screening effect due to hydrogen bonding of an otherwise "accessible" histidine. A significant corollary to this finding would be that the catalytic histidine is rigidly entrapped in a remarkably strong hydrogen-bonding network, a situation that may pertain to mechanistic aspects of catalysis.
Berna
P P
PP
Laboratoire d'Interactions Moléculaires et de Technologie de Séparation, Université de Technologie de Compiègne, URA-CNRS 1442, France.
Mrabet
N T
NT
Van Beeumen
J
J
Devreese
B
B
Porath
J
J
Vijayalakshmi
M A
MA
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002614:Chelating Agents / Q000378:metabolism; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D002919:Chymotrypsinogen / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007098:Imino Acids / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D013499:Surface Properties
D002614:Chelating Agents; D007098:Imino Acids; D006639:Histidine; D003300:Copper; D002919:Chymotrypsinogen; D002918:Chymotrypsin; C008109:iminodiacetic acid