Reaction Rates at Enzyme Active Sites

Measurement of Reaction Rates at Enzyme Active Sites 

When enzyme is first mixed with substrates (in excess over the enzyme), one can often observe a burst of product formation at a rate faster than steady-state turnover. This presteady-state burst is due to the accumulation of product at the active site of the enzyme. On quenching the reaction mixture with a denaturant to stop further reaction, the enzyme-bound product is liberated and the quantitation of product includes the sum of that bound to the enzyme and free in solution at the time of the quench. 

Burst kinetics are often described in terms of a two-step irreversible mechanism (5, 38). 

The equation describing the time dependence of formation of product will be given by an exponential followed by a linear phase 

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). 

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V. Measurement of Reaction Rates at Enzyme Active Sites. 36... 

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