Presteady-state burst kinetics

Figure 13 indicates burst kinetics. As discussed before, such biphasic curves indicate the reaction to occur through two steps involving an acylated intermediate. The initial slopes for the presteady state can be taken as the measure of acylation rates, and the slopes of the later straight line for steady-state can be taken as the measure of deacylation rates. 

The presteady-state burst will be followed by steady-state turnover at a rate given by cat The presteady-state burst of product formation will occur at a rate defined by the sum of the rates of the chemical reaction and product release. The amplitude is also a function of both rate constants, k2 and kj. Thus, the amplitude of the burst can be predicted from the rate of the burst and the rate of steady-state turnover. Although this model can account for burst kinetics, it is often inadequate due to the assumed irreversibility of the chemical reaction. The internal equilibrium arising from the reverse of the chemical reaction k-2) reduces the amplitude of the burst to less than predicted by Eq. (26). 

Because of the factors that reduce the amplitude in a presteady-state burst experiment and the difficulty in resolution of the product (or intermediates) from excess substrate, it is often desirable to use single-turnover methods. These experiments are performed with enzyme in excess over substrate to allow the direct observation of the conversion of substrates to intermediates and products in a single pass of the reactants through the enzymatic pathway. Unlike the presteady-state burst experiments, the kinetics are free of complications resulting from the steady-state formation of products, which limits the resolution of the burst kinetics and the detection of any intermediates above the background of excess substrates and products. 

Fig. 7. Dynein ATPase burst kinetics. The kinetics of a presteady-state burst of ATP binding (o) and hydrolysis ( ) were determined at two ATP concentrations (A) 30 and (B) 50 /xAf. The data fit rate constants of k = 4.7 juAf sec", ki = 55 sec, k-2 = 10 see", and ks = 8 see" according to Scheme IV. Reproduced with permission from (J9).

Presteady-state burst kinetics can be fit to an equation of the form... 

Information extracted from kinetic data collected under burst conditions, in which there is a two- to fourfold excess of DNA substrate over DNA polymerase, illustrates another important application of presteady-state experiments. This type of experiment provides useful information about the transient concentration of kinetically active ternary complex. A time course of DNA product formation under these conditions demonstrates a transient exponential phase followed by a steady-state linear phase. By examining the dependence of the burst amplitude on DNA concentration, the enzyme s binding affinity for DNA can be evaluated. 

By means of Eqn. 17 and stopped-flow studies at various values of Sq, k2, and kj, can be separately determined and studied. For carboxypeptidase Y, which also shows such burst kinetics with 4-nitrophenyl trimethylacetate (I), enzyme preparations with differing amounts of attached carbohydrate, reacted with closely similar steady-state parameters K, k ) but differences were apparent for presteady-state parameters (k2> [14]. 

NADH or aldehyde) in excess of the other reactant. Under these conditions, the chemical conversion of aldehyde to alcohol occurs with a (saturated) apparent first-order rate constant of 200 to 400 sec i. This process, as measured either by the disappearance of NADH or by the disappearance of chromophoric aldehyde, has been shown by McFarland and Bernhard (80) to be subject to a primary, kinetic isotope effect ksjkj) =2 to 3 when stereospecifically labeled (4-R)-deuterio NADH is compared to isotopically normal NADH. Shore and Gutfreund (84) earlier had investigated substrate kinetic isotope effects on the pre-steady-state phase of ethanol oxidation. Their studies demonstrated that the rate of the presteady-state burst production of NADH is subject to a primary kinetic isotope effect, kulkj) 4—6 when 1,1-dideuterio ethanol is compared to isotopically normal ethanol, and that there is no primary kinetic isotope effect on the steady-state rate. It can be concluded from these studies (a) that the rate of interconversion of ternary complexes e.g., Eq. (19) above], as already mentioned, is rapid relative to turnover, and (b) that the transition-state for the rate-limiting step in the interconversion of ternary complexes involves carbon-hydrogen bond scission and/or carbon-hydrogen bond formation. 

---