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 w ater molecule also held by residues Glu168 and Glu211. The water molec ule is suggested to participate in the catalytic mechanism (Lebioda, L . and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His3 73 with asparagine (H373N enolase) or phenylalanine (H373F enolase) re duces enzymatic activity to ca. 10% and 0.0003% of the native enzyme a ctivity, respectively. H373N enolase exhibits a reduced K-m for the su bstrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP(1), relative to n ative enolase. H373F enolase binds AEP less strongly, producing a smal ler absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrat e; H373N enolase subunit dissociation is less than H373F enolase but m ore than native enolase. Substrate and Mg2+ increase subunit associati on in both mutants. Differential scanning calorimetric experiments ind icate that the interaction with substrate that stabilizes enolase to t hermal denaturation involves His373. We suggest that the function of H is373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O s ystem, which produces removal or addition of hydroxyl at carbon-3 of t he substrate. (C) 1997 Elsevier Science B.V.