Role of His159 in yeast enolase catalysis

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enola se from yeast and lobster muscle, However, the identities of the residues f unctioning as the general acid/base pair are not yet established unambiguou sly. In the Mn2+-phosphoglycolate complex of lobster muscle enolase, the im idazole group of His 157 (His 159 in the yeast enolase numbering system) is in van der Waals contact (4.5 Angstrom) with the C-2 of the inhibitor [Duq uerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further informa tion about the role played by His159 in the catalytic mechanism of yeast en olase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indica ted by near- and far-UV circular dichroism and fluorescence data, and the m utation has no significant effect on the formation of ternary and quaternar y enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wildty pe specific activity, which corresponds to a reduction in k(cat) of 4 order s of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His 159 serving as a potential catalytic base in the enolase reaction. We have suggested that Hi s159 could also serve as a metal ligand at the third, inhibitory, metal bin ding site. This proposal is consistent with the catalytic mechanism of-yeas t enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C-2 proton from 2-PGA, Metal binding studies support the above proposal. Mn2+ binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn2+ at the third, inhibitory site of H159A is a factor of 3 weaker compare d to wild-type enolase. The factor of 3 in binding is reasonable for the co ntribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100, Regan, L. ( 1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281, Cha et al, (1994) J. Biol. Chem. 269, 2687 - 2694].