Abstract

Enolase is an essential enzyme in glycolysis and gluconeogenesis. In 2001, the first known case of a human disorder diagnosed as muscle enolase deficiency was found (Comi et al., 2001). The patient carried two heterozygous missense mutations changing glycine 156 to aspartate 156 in one case and glycine 374 to a glutamate 374 in the other. In this study, yeast enolase is used as a model to study the role of G156 (G157 in yeast enolase) and the effects of the G156D mutation in human ββ-enolase. Specifically, site directed mutagenesis was used to change G157 to D in yeast enolase. Spectroscopic studies using fluorescence, circular dichroism, and analytical ultracentrifugation indicated no substantial differences in the conformational foldings of G157D compared to wild-type enolase. The conformational stability of the G157D variant was found to be slightly decreased compared to the wild-type enolase when subjected to chemical or thermal denaturation. The G157D variant is also more susceptible to limited proteolysis by trypsin. The G157D protein exhibits only 0.06% wild-type activity under standard enolase assay conditions. Kinetic studies indicate that the G157D enolase has a higher K M for Mg 2+ , no significant differences in K M for Mn 2+ , and a slightly increased K M for its substrate, 2-phospho-D-glycerate, than has the wild-type enzyme. 2 H kinetic isotope effect studies proved that the first step of enolase catalysis, abstraction of the C-2 proton from 2-PGA becomes more rate limiting for the G157D enolase. Ligand binding studies indicate similar K d for the first Mg 2+ site for both G157D and wild-type enolase. Isothermal titration calorimetry indicates comparable association constants for the second Mn 2+ , PhAH, and 2-PGA/PEP. To further investigate the effects of size and charge upon replacing a small, non-charged glycine residue by a bulky, charged aspartate, G157A, G157N, and G157L yeast enolase variants were also created. Studies on the latter three proteins illustrate correct folding of the proteins and no substantial stability discrepancies compared to the wild-type enzyme. In spite of this, activity studies show that as the size of the amino acid at this site increases, the enzyme activity decreases. It is possible that the small and non-charged properties of G157 are necessary for proper coordination of active site residues to the metal cofactors and the substrate 2-phospho-D-glycerate