Dehydrogenases velocity constants

The initial rate equation is again of the form of Eq. (1) with the kinetic coefficients as in Table I, which shows that the mechanism differs from the simple ordered mechanism in three important respects. First, the isomerization steps are potentially rate-limiting evidence for such a rate-limiting step not attributable to product dissociation or the hydride-transfer step (fc) has been put forward for pig heart lactate dehydrogenase 25). Second, Eqs. (5) and (6) no longer apply in each case the function of kinetic coefficients will be smaller than the individual velocity constant (Table I). Third, because < ab/ a< b is smaller than it may also be smaller than the maximum specific rate of the reverse reaction that is, one of the maximum rate relations in Eq. (7) need not hold 26). This mechanism was in fact first suggested to account for anomalous maximum rate relations obtained with dehydrogenases for which there was other evidence for an ordered mechanism 27-29). 

Velocity Constants fob Enztme-Coenzyme Reactions of Allostbbic Dehydrogenases... 

The results of stopped-flow studies of the lactate dehydrogenase reaction have proved to be more difficult to interpret than those of alcohol dehydrogenase. The dissociation velocity constants for the binary NADH compounds of the pig heart and skeletal muscle lactate dehydrogenases are much larger than that for liver alcohol dehydrogenase, and also larger than the maximum specific rates of lactate oxidation at pH 6.0-7.0 (Table VII). Some earlier step must therefore be rate-limiting. 

Km is the Michaelis constant. In some cases such as hydrolases or lactic dehydrogenases (T2), the velocity may fall again with higher substrate concentrations, so that there is an optimum substrate concentration which approximates the theoretical value V, the maximal velocity, following the theory of Michaelis and Menten... 

Other 4-nitrophenyl esters have also been reported to be substrates of various hydrolases. For example, 4-nitrophenyl hexanoate (7.19) was hydrolyzed by bovine serum albumin [39], The affinity of the substrate for the macromolecule was found to be high (Km/n = 0.040 mM, where n is the number of sites), but the reaction itself was slow ( = 5 10-3 s-1, where k2 is the first-order rate constant of the formation of the phenol product from the enzyme-substrate complex). Another ester, 4-nitrophenyl pivalate (7.20), was hydrolyzed by cytoplasmic aldehyde dehydrogenase at a maximum velocity ca. 1/3 and an affinity ca. 1/20 those of the acetate [40], However, the rate-limiting steps were different for the two substrates, namely acylation of the enzyme for the pivalate, and acyl-enzyme hydrolysis for the acetate (see Chapt. 3). 

The best-known exception to exponential kinetics is the elimination of alcohol (ethanol), which obeys a linear time course (zero-order kinetics), at least at blood concentrations > 0.02 %. It does so because the rate-limiting enzyme, alcohol dehydrogenase, achieves half-saturation at very low substrate concentrations, i.e at about 80 mg/L (0.008 %). Thus, reaction velocity reaches a plateau at blood ethanol concentrations of about 0.02 %, and the amount of drug eliminated per unit of time remains constant at concentrations above this level. 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

The isomerization of a transitory conplex does not affect the algebraic form of the velocity equation in the absence or in the presence of products, but the composition of some kinetic constants are changed by introducing the rate constants for isomerization k and jtff in the forward dii on and and k in the reverse direction. The isomerization of transitory complexes is very common in many enzyme reactions, particularly among pyridine-dependent dehydrogenases. 

Fig. 2. Reaction of 3 -p-fluorosulfonylbenzoyladenosine with bovine liver glutamate dehydrogenase. Glutamate dehydrogenase (021 mg/ml) was incubated with 3 -FSBA (0.496 mil/) at 24° in 0.01 M sodium barbital buffer (pH 8) containing 0.43 M KCl and 5% ethanol. At each indicated time, an aliquot was withdrawn, diluted 20-fold with Tris-0.1 M acetate buffer (pH 8) at 0°, and assayed (A) in the absence and (B) in the presence of 100 yM ADP. Inset Determination of the pseudo first-order rate constant from the decrease in activation by ADP. (Ft and Fo are the enzymic velocities measured in the presence of ADP and the given and zero time, respectively, and F > is the constant velocity at the end of the reaction. The pseudo first-order rate constant calculated is 0D351 min. ) Data are taken from P. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).

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