Abstract

Enolase, a dimeric enzyme, in the presence of Mg$\sp{2+}$ catalyzes the reversible dehydration of 2-phospho-D-glycerate (2PGA) to form phosphoenolpyruvate (PEP) in the glycolytic pathway. Studies in the inactivation of rabbit muscle enolase by NaClO$\sb4$ show that both Mg$\sp{2+}$ and 2PGA protect the enzyme from the inactivation. The protection provided by Mg$\sp{2+}$ and 2PGA is attributed to a series of interactions between the ligands and the active site residues, and to the interactions throughout the side chains of the secondary elements in the two monomers. However, surprisingly, in the absence of Mg$\sp{2+}$, 2PGA still protects the apo-enolase from inactivation even at high concentrations of metal chelating reagents such as EDTA and EGTA. This is not supposed to occur because, according to the literature, the substrate binds to the enzyme only when a divalent metal ion is bound in the high affinity site I. Inductively coupled plasma mass spectroscopy was applied to the apo-enolase sample to identify the trace metal ions, but those trace metal ions are not responsible for the protection of the apo-enolase by 2PGA. The effects of several inhibitory substrate analogues, phosphoglycolate, phosphonoacetic acid, 3PGA and glycerol 2-phosphate, were characterized. (Abstract shortened by UMI.)