Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site

Abstract

Exchange-inert β,γ-bidentate Cr(H 2O) x(NH 3) yATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min −1 to 0.118 min −1 for Cr(NH 3) 4ATP and Cr(H 2O) 4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k obs by 3-PGA was proportional to the number of NH 3 ligands present in the coordination sphere of Cr 3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr 3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H 2O) 4ATP. Δ diastereomer of Cr(H 2O) 4ATP yielded an order of magnitude smaller K i value compared to the value observed with the Λ isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H 2O) 4ATP or Cr(NH 3) 4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr 3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme ·CrATP and enzyme·CrATP·3-PGA complexes.