Malic Enzyme of Escherichia coli

Abstract

Abstract A kinetic study of partially purified malic enzyme from Escherichia coli was made. The double reciprocal initial velocity plots of both of the substrates, TPN+ and malate, were linear in the absence of allosteric inhibitors (oxalacetate, acetyl coenzyme A, and DPNH). Product inhibition studies with bicarbonate and pyruvate gave generally only linear inhibitions with either TPN+ or malate as varied substrates. With TPNH as the inhibitor, however, the initial velocity plots for malate became nonlinear. This nonlinearity vanished when assays were performed in the presence of 0.72 m glycine, a reagent which desensitizes the enzyme to various allosteric effectors. In the presence of this desensitizing agent TPNH gave linear noncompetitive inhibition against TPN+ as the varied substrate. From these studies it was postulated that malic enzyme followed an ordered mechanism with isomerization of the free enzyme as an obligate step of the reaction pathway. In the presence of each of the allosteric inhibitors, the initial velocity plots for TPN+ remained linear but the plots for malate became nonlinear. Inhibition by acetyl-CoA and oxalacetate was competitive with malate, but noncompetitive with TPN+. The kinetic behavior of malic enzyme in the presence of allosteric inhibitors was analyzed in terms of a two conformational state model and the conclusions reached were (a) that the E-TPN+ complex existed in the absence of allosteric ligands in a form which had a high affinity for malate (State R) and (b) that the binding of any one of the several inhibitors to the enzyme resulted in its conversion to a state (T) which had very little affinity for malate.