Substrate activation by malate induced by oxalate in the Ascaris suum NAD-malic enzyme reaction

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Abstract
Substrate activation of the rate of the NAD-malic enzyme reaction by malate is obtained in the presence but not in the absence of oxalate. The substrate activation is a result of competition between malate and oxalate for the E .cntdot. NADH complex, with malate binding to the form of the complex unprotonated at an enzyme group with a pK of 4.9 and oxalate binding preferentially to the protonated form. The off-rate for NADH from the E .cntdot. NADH complex is completely rate limiting when the group with a pK of 4.9 is protonated but is only one of several rate-limiting steps when it is unprotonated [Kiick, D. M., Harris, B. G., and Cook, P. F. (1986) Biochemistry 25, 277]. The competition by malate with oxalate thus results in an overall increase in the off-rate for NADH as a result of binding to the unprotonated from E .cntdot. NADH. Consistent with the proposed mechanism, the deuterium isotope effect on V for the nonsubstrate-activating malate concentration range decrease from 1.6 in the absence of oxalate to 1.3 in the presence of a concentration of oxalate equal to its Kii. The rate equation for the oxalate-induced substrate activation by malate is derived and presented in the Appendix. Data are discussed in terms of the overall mechanism of the NAD-malic enzyme.