Inhibition of Creatine Kinase by Chromium Nucleotides

Abstract

Abstract The steady state kinetics of creatine kinase have been studied at pH 7 by initial velocity, product inhibition, and dead end inhibition techniques using chromium ATP and chromium ADP. The mechanism appears to be rapid equilibrium random in the MgADP plus phosphocreatine direction and equilibrium ordered with MgATP adding before creatine in the other direction. This degenerate form of the rapid equilibrium random mechanism found in both directions at pH 8 may be caused by protonation of a group on the enzyme at pH 7 that will not permit binding of the positively charged portion of creatine until the MgATP is already bound. The chromium nucleotides were competitive inhibitors versus the magnesium nucleotides and noncompetitive versus the guanido substrates. Except for MgATP, chromium and magnesium nucleotides have about the same affinity for the enzyme at pH 7. The 10-fold greater affinity of CrATP over MgATP for the enzyme may reflect an equilibrium between doubly (β,γ) and triply (α,β,γ) coordinated MgATP in solution with the triply coordinated nucleotide being the actual substrate. Nitrate synergizes creatine inhibition of reaction in the MgADP plus phosphocreatine direction, but was ineffective in synergizing CrADP inhibition in the MgATP plus creatine direction. This may be because the planar PO3 group which nitrate mimics in the E-MgADP-creatine-NO3 complex is coordinated to the Mg2+, but while nitrate can displace the water which is replacing PO3 from Mg2+, it cannot displace the water molecule covalently coordinated to chromium in the corresponding E-CrADP-creatine complex.