Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

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Abstract
Malic enzyme of pigeon liver binds NADPH at 4 equivalent enzyme sites and binds Mn2+ and malate each at 2 sets of tight and weak sites with negative cooperativity. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn22+-NADPH4, E4-Mn22+-NADPH4-dimalate, and E4-Mn22+-NADPH4-tetramalate by large excess NADP or its analog phosphoadenosine(2'')diphospho(5'')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn22+-NADPH4 complex containing 2 tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by 2 simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k .simeq. 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports the proposed half-of-the sites model. Dissociation from the E4-Mn22+-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at 2 sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at 2 additional weak sites yields E4-Mn22+-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition. The slow onset of substrate inhibition is a first-order process consistent with a slow malate-induced conformational change of the quaternary complex from an active to a less active form.