title: Role of His159 in yeast enolase catalysis.
abstract: There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase 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 indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast 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. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution 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].
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Vinarov
authors.forename: T
authors.initials: T
authors.lastname: Nowak
mesh_terms: D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006016:Glycolates / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010728:Phosphoenolpyruvate / Q000737:chemistry; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D006016:Glycolates; D008024:Ligands; D046911:Macromolecular Substances; D011522:Protons; D011994:Recombinant Proteins; D008345:Manganese; D006639:Histidine; D010728:Phosphoenolpyruvate; D010751:Phosphopyruvate Hydratase; C013193:phosphoglycolate; D000409:Alanine