PROTON-TRANSFER BY HISTIDINE-67 IN SITE-DIRECTED MUTANTS OF HUMAN CARBONIC-ANHYDRASE-III

The ability of a histidine residue at position 67 in human carbonic an hydrase III to transfer protons in the catalytic pathway for the hydra tion of CO2 was investigated for a series of site-specific mutants. Wi ld-type carbonic anhydrase III has an arginine at this position with t he C alpha of residue 67 about 9.4 Angstrom from the zinc. The active- site cavity contains no other residues capable of facile proton transf er. Rate constants for proton transfer from His 67 to the zinc-bound h ydroxide were determined from the rate constants for the exchange of O -18 between CO2 and water measured by mass spectrometry. A range of va lues for the pK(a) of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to t he zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1. 3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding u nfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen , X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemi stry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work func tion) that probably represents an energy requirement for proper alignm ent of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier a ppears the same for His 67 or His 64.