Arguments are presented which indicate that the low steady-state rates of citrate production governing the catalytic interaction of citrate synthase from pig heart with citryl-CoA reflect the formation of a non-productive enzyme . citryl-CoA complex. The kinetic predictions of such an extended reaction mechanism are examined and are shown to account in satisfactory detail for the complex multiphasic rate behaviour exhibited by the enzyme under a variety of conditions in reactions involving citryl-CoA as a substrate.Citrate synthase catalyses the formation of citrate and CoA from oxaloacetate and acetyl-CoA in the citric acid cycle. Kinetic and equilibrium binding studies have established that the enzyme operates by a sequential mechanism [l, 21 such that both substrates must be bound before any reaction products are released. This observation indicates that the catalytic reaction may proceed via intermediate formation of citrylCoA. Conclusive evidence in the same direction comes from the demonstration that the interaction of citrate synthase with citryl-CoA leads to rapid formation of citrate at a rate which is initially comparable to that of the physiological reaction involving oxaloacetate and acetyl-CoA [3].Reactions initiated with citryl-CoA as the substrate exhibit some unexpected kinetic characteristics, however. Thus, the initial phase of rapid citrate formation is transient and followed by a steady-state phase of product formation at a maximum rate which is 50-fold lower than that of the physiological reaction. A third reaction phase, characterized under certain conditions by initially increasing and finally decreasing rates of citrate formation, can be distinguished at the end of the reaction when the consumption of citryl-CoA approaches completion. This multiphasic kinetic pattern has been proposed to depend on the reactivity of the physiological substrates formed from citryl-CoA on reversal of the synthase condensation step. Acetyl-CoA and oxaloacetate not only accumulate during reactions initiated with citryl-CoA but are simultaneously utilized for citrate production by the physiological pathway. A flow equilibrium between physiological substrates and citryl-CoA was envisaged to obtain during the transient phase, thus yielding non-productive enzyme and the low rate of citrate formation during the steady-state phase [4].We have attempted to simulate the kinetics of enzymic citrate formation from citryl-CoA on basis of that proposal. The results indicated that the existence of a flow equilibrium involving the physiological substrates may provide a reasonable explanation for certain characteristics of the catalytic Kemicentrum, Lunds Universitet, Box 124, S-22100 Lund, Sweden reaction, but the low steady-state velocities observed with citryl-CoA as the substrate cannot be accounted for in such terms. In the present investigation, an extended reaction mechanism is put forward which attributes the low rate of citryl-CoA turnover to the formation of a non-productive enzyme citryl-CoA complex. The reaction ...

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1. The kinetics of inhibition of citrate synthase by a number of dicarboxylic and tricarboxylic acids have been examined. Inhibition parameters providing information about equilibrium constants for ligand-binding to free enzyme and to the binary enzyme . oxaloacetate complex were determined under Michaelis-Menten conditions. Ligand-binding to the binary enzyme . acetyl-CoA complex was studied by a new kinetic approach, based upon an examination of the effect of inhibitors on the second-degree rate behaviour of the enzyme.2. Carboxylic acids may bind to the acetyl-CoA site (as anions), as well as to the oxaloacetate site (as oxaloacetate analogues). Depending on the relative strength of these interactions, inhibition competitive with either or both of the substrates is observed.3. Kinetically estimated equilibrium constants for the binding of inhibitors to different enzyme species in the catalytic mechanism are reported. The results obtained show that carboxylic acids, in general, bind non-cooperatively with the substrates.4. The affinity of citrate synthase for acetyl-CoA increases about 10-fold on the binding of D-malate. The mechanistic significance of this and previously reported heterotropic cooperativity effects in the citrate synthase system is discussed. It is suggested that the binding of oxaloacetate and certain carboxylic acids containing a D-malate substructure induces a conformational change of the enzyme. This conformational change serves the purpose of creating a binding site for proper accomodation of the acyl moiety of acetyl-CoA at the catalytic center of the enzyme.Citrate synthase catalyzes the condensation reaction between acetyl-CoA and oxaloacetate in the citric acid cycle. The enzyme operates by a basically random ternary-complex mechanism (Scheme 1) [I -41. Michaelis-Menten kinetics are obeyed at low substrate concentrations, and under such conditions the catalytic reaction proceeds by an effectively ordered mechanism with oxaloacetate adding first to the enzyme [5,6]. High concentrations of acetyl-CoA cause deviations from Michaelis-Menten kinetics, typical of substrate inhibition, in the reaction catalyzed by the pig-heart enzyme [4]. This kinetic pattern was recently shown to be due to intervention of the alternative pathway for ternary-complex formation in which acetyl-CoA binds first to the enzyme [7]. Comparison between rate constants in the two pathways to the ternary enzyme-substrate complex led to the conclusion that the affinity of citrate synthase for either substrate increases at least 20-fold on the binding of the second substrate [7], i.e. there is a strong heterotropic cooperativity in the process of substrate binding to the enzyme.