J. Biol. Chem., Vol. 263, Issue 24, 11704-11710, Aug, 1988
Proton/product time course ratios: a new approach to transient-state kinetic analysis
HF Fisher, S Maniscalco, N Singh and SA Adediran
Department of Biochemistry, University of Kansas School of Medicine, Kansas City 64128.
The usefulness of the indicator dye method for the detection of transient
enzyme-product intermediates has been very limited due to the near
impossibility of resolving the apparent single exponential time courses
resulting from sequences of steps linked by rather similar rate constants.
We propose here a novel approach, the proton-product time course method, a
procedure which can extract a great deal of the mechanistic information
which remains buried in conventional proton release-time course
measurements. The method involves nothing more than measuring the ratio, r,
of the moles of H+ released to the moles of product formed as a function of
time. We derive the theory relating this r function to mechanisms of
varying complexity, explore the theoretical behavior of the function in
various possible mechanistic situations, and employ the new approach in an
experimental system. We demonstrate the fact that the proton/product time
course ratio method can provide evidence of the existence of hidden steps
in transient state kinetic studies, that it can determine accurate
thermodynamic pK values of the intermediate complexes involved in those
steps, and that it can produce time courses of individual intermediates
which are obscure to conventional kinetic methods.
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