Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties.
The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.
Brewer
J M
JM
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Glover
C V
CV
Holland
M J
MJ
Lebioda
L
L
D001216:Asparagine; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005988:Glyceric Acids / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D006358:Hot Temperature; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D011773:Pyruvates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013646:Tartronates / Q000378:metabolism
D005988:Glyceric Acids; D011773:Pyruvates; D013646:Tartronates; C027028:tartronate semialdehyde; C008885:2-phosphoglycerate; C027322:3-aminoenolpyruvate-2-phosphate; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D010751:Phosphopyruvate Hydratase; D008274:Magnesium