Fumarase kinetics

In the Briggs-Maldane mechanism, when k2 is much greater than k-i, kcJKM is equal to kx, the rate constant for the association of enzyme and substrate. It is shown in Chapter 4 that association rate constants should be on the order of 108 s l M l. This leads to a diagnostic test for the Briggs-Haldane mechanism the value of kaJKu is about 107 to 108 s-1 M-1. Catalase, acetylcholinesterase, carbonic anhydrase, crotonase, fumarase, and triosephosphate isomerase all exhibit Briggs-Haldane kinetics by this criterion (see Chapter 4, Table 4.4). 

As an application of equation 7.6-5, consider the effect of pH on the inhibition constants of fumarase, which have been determined for succinate, D-tartrate, L-tartrate, and meso-tartrate inhibitors (Wigler and Alberty, 1960). The kinetics of the conversion of fumarate to L-malate and the inhibition by these competitive inhibitors indicate that there are two acid groups in the catalytic site that affect the binding ... 

To understand how the TCA cycle responds kinetically to changes in demand, we can examine the predictions in time-dependent reaction fluxes in response to changes in the primary controlling variable NAD. Figure 6.4 plots predicted reaction fluxes for pyruvate dehydrogenase, aconitase, fumarase, and malate dehydrogenase in response to an instantaneous change in NAD. The initial steady state is obtained... 

Brandt DA, Barnett LB, Alberty RA. The temperature dependence of the steady state kinetic parameters of the fumarase reaction. J. Am. Chem. Soc. 1963 85 2204-2209. 

There is still a third possible mechanism for the fumarate hydratase reaction. The proton and hydroxyl groups may be added simultaneously in a concerted reaction. However, observed kinetic isotope effects are not consistent with this mechanism. In 1997 the structure of fumarase C of E. coli was reported. Each active site of the tetrameric enzyme is formed using side chains from three different subunits. The H188 imidazole is hydrogen bonded to an active site water molecule and is backed up by the E331 carboxy-late which forms a familiar catalytic pair. However, these results have not clarified the exact mode of substrate binding nor the details of the catalytic mechanism. Structural studies of fumarate hydratase from yeast and the pig are also in progress. 

In general, enzymes exhibit maximum catalytic activity at a definite pH. This optimum pH is generally in the vicinity of pH 7 ( 1), although exceptions are well known. The dependence of the kinetic parameters of the fumarase reaction on pH is illustrated in Figs. 9-2 and 9-3. Several different possible effects of pH on the reaction must be distinguished. In the first place, many substrates may have ionizable groups and only one of the ionized forms of the substrate may be acted upon by the enzyme. Since substrate ionization constants can be determined quite easily and precisely,... 

An excellent study has been made of the temperature dependence of the steady-state kinetic parameters of the fumarase reaction [6]. By studying the temperature dependence over a wide range of pH, the apparent activation energies and standard-enthalpy changes associated with the pH-independent steady-state parameters, the lower bounds of the rate constants, and the ionization constants of the groups at the active site were obtained. The results are summarized in Table 9-1. In this case the temperature dependence of all parameters appears quite normal. The standard-enthalpy changes of... 

Active immobilized cells investigation of 660 kinetics and decay of fumarase activity of the immobilized cells... 

As an example, consider a dehydration of L-malate catalyzed by fumarase, affording fumarate as the product of reaction this reaction is fully reversible. Kinetic studies suggest a model in which a histidine residue and a carboxyl group in the active site of enzyme are necessary for catalysis. According to this model, from the malate side of reaction, the histidine must be protonated and the carboxyl ionized, while in the reverse direction, the states of ionization are reversed (Cleland, 1977) (Fig. 11). Note that, in Fig. 11, proton is not a part of the chemical reaction but, formally, the elements of water (proton and the hydroxyl ion) are removed from the substrate by the enzyme. 

Based on these results, Goldberg et al. (2006) suggested two possibilities to explain these findings. The first is that in R. oryzae the cytosolic fumarase is kinetically different from the mitochondrial isoenzyme, due to distinct posttranslational modifications, or to specific conditions in the two compartments. The second possibility is that R. oryzae harbors two genes encoding two different fumarases, one in mitochondria, which catalyzes the conversion of fumaric to L-malic acid, and a cytosolic enzyme, which catalyzes the conversion of L-malic to fumaric acid. Upon transfer into medium C, a fumarase with unique characteristics is induced. L-malic acid s conversion to fumaric acid is enhanced by the induced fumarase and when the concentration of fumaric acid in the cell exceeds 2 mM, the reverse reaction to L-malic acid is fully inhibited. Thus, this property of the unique fumarase, whose existence was then only hypothesized, can ensure that fumaric acid is accumulated. 

Kinetics of Fumarase Activity. The kinetics of the fumarase reaction have been studied intensively by Alberty and his collaborators. They have found that interaction of enzyme with phosphate can cause activation at low phosphate concentrations, but that at high concentrations, phosphate acts as a competitive inhibitor. An unusual effect was noted when the effect of fumarate concentration on the rate of hydration was measured. At low substrate concentrations the Lineweaver-Burk plots are linear, but at higher concentrations the rate is faster than anticipated. This phenomenon was interpreted as indicating an interaction of fumarate with the enzyme at sites other than the catalytic site, to form a more active enzyme. At very high substrate concentrations (0.1 M) there is inhibition of the reaction, and the theoretical V— is never attained. 

Isotopic Studies with Fumarase. The kinetic data reported above have led to preliminary attempts to interpret the nature of the binding of substrate to enzyme in terms of specific amino acid side chains. Another approach to the nature of the reaction was made in studies with D20. It was found that the hydrogens of fumarate do not equilibrate with... 

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