Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies

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Abstract
Isotope effects were measured with Mg2+ as the activator and L-malate labeled with 2H or 3H at C 2 as the substrate [for pigeon liver malic enzyme] over the pH range 4-10. Comparison of the nearly pH-independent 2H-isotope effect on V[velocity]/Kmalate of 1.5 with the 3H effect of 2.0 by the method of Northrop gives limits on the true effect of 2H substitution on the bond-breaking step of 5-8 in the forward reaction and 4-6.5 in the reverse direction. Comparison of the 2H effect on V/K with the 13C-isotope effect of 1.031 reported by Schimerlik et al., allows the deduction that at pH 8 reverse hydride transfer is 6-8 times faster than decarboxylation, which is thus largely rate limiting for the catalytic reaction. The absence of a 2H-isotope effect on V at pH 7-8 and comparison of the Ki [inhibition constant] of pyruvate as an uncompetitive inhibitor of the forward reaction and a substrate for the reverse reaction indicate that at neutral pH the release of NADPH from enzyme-NADPH. (E-NADPH) is the rate-limiting step in the forward direction. The observation of a 2H effect on V that approaches 3 at pH 4 and 10 shows that, at very low and very high pH, hydride transfer may become partly rate limiting. In the reverse reaction, the probable rate-limiting step at pH 7 is the isomerization of E-NADPH, while at pH 8.5 and above V becomes too large to measure and appears infinite. Substitution of Co2+, Ni2+ or low levels of Mn2+ for Mg2+ gives similar 2H-isotope effects, although the values of V and Kmalate vary considerably with metal. The kinetics of Mn2+ show pronounced negative cooperativity, with Km values of 7 .mu.M and 5 mM for concentration ranges from 4-100 .mu.M and over 1 mM.

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