Enzymes presteady-state burst kinetics

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. 

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]. 

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