STRUCTURE OF THE BIS DIVALENT-CATION COMPLEX WITH PHOSPHONOACETOHYDROXAMATE AT THE ACTIVE-SITE OF ENOLASE

Phosphonoacetohydroxamate (PhAH) is a tight-binding (K(i) = 15 pM) inhibitor of enolase that is believed to mimic the aci-carboxylate form of the intermediate carbanion in the reaction [Anderson, V. E., Weiss, P. M., & Cleland, W. W. (1984) Biochemistry 23, 2779]. Electron paramagnetic resonance (EPR) spectroscopy of Mn2+ has been used to map sites of interaction of PhAH with the two divalent cations at the active site of enolase from bakers' yeast. EPR spectra of enolase-PhAH complexes containing two Mn2+ bound at the active site contain multiple fine structure transitions each with a 45-G Mn-55 hyperfine spacing that is a characteristic of spin exchange coupled pairs of Mn2+. Magnetically dilute complexes were obtained by preparation of specific Mg2+/Mn2+ hybrid complexes by manipulating the order of addition of the divalent metal species. Thus, Mn2+ was placed in the higher affinity site by addition of 1 equiv of Mn2+ to a solution of enolase and PhAH, followed by addition of 1 equiv of Mg2+. Reversing the order of addition of Mg2+ and Mn2+ placed Mn2+ in the lower affinity site. Regiospecifically O-17-labeled forms of PhAH were prepared, and the binding of the functional groups on PhAH to Mn2+ at the two metal ion sites was determined from the presence or absence of O-17 superhyperfine coupling in the EPR signals. The hydroxamate oxygen is a ligand of Mn2+ at the higher affinity site, a phosphonate oxygen is a ligand of Mn2+ at the lower affinity site, and the carbonyl oxygen is a mu-O bridge of the two metal ions. The binuclear chelate structure of the bound inhibitor suggests electrophilic roles for both divalent cations in stabilization of the aci-carboxylate form of the intermediate carbanion as well as an electrophilic role of the high-affinity metal ion in augmenting the leaving group ability of the 3-OH of 2-phospho-D-glycerate.