Product inhibition in mechanisms in which the free enzyme isomerizes.

Product inhibition in mechanisms in which the free enzyme isomerizes In any enzyme mechanism, the form of free enzyme left after the last product has been released may be different from the one to which the first substrate binds, so that the catalytic cycle is completed by an enzyme isomerization to regenerate the original form. Efforts to detect such isomerization met with little success, however, until the introduction of the tracer perturbation technique by Britton (1966, 1973), which was used by him and his associates to study a number of mutases; more recently it has been applied to proline racemase (Fisher et al., 1986), triose phosphate isomerase (Raines and Knowles, 1987), and fumarase (Rebholz and Northrop, 1993). Britton and associates refer to the technique as 'induced transport', but the term 'tracer perturbation' proposed by Albery and Knowles (1987) gives a clearer indication of what it involves, and will be used here. In principle, free-enzyme isomerization can also be detected by product inhibition, because it generates a term in ap in the denominator of the rate equation, where a and p are the concentrations of first substrate and last product respectively. It is obvious that if the isomerization is very fast this term will be negligible, as has long been realized attention to the less obvious point that it also becomes negligible when the isomerization is slow, so that product inhibition cannot provide a reliable indication of whether a kinetically significant isomerization occurs. Rebholz and Northrop (1993) have recently contested this analysis, claiming that 'Britton (1973) was frustrated by the lack of a definite relationship between his kinetic constants and the relative rates of isomerizations', and explaining this hypothetical frustration in terms of two supposed errors in his analysis. In a note added in proof, they add that 'Britton's intuition that inhibition will appear competitive in the presence of slow isomerization appears to be correct', but they do not explain why they ascribe this conclusion to Britton's intuition rather than to his algebraic analysis, nor why they thought it useful to devote several pages to a laboured account of their efforts to arrive at an opposite (and wrong) conclusion. Here I show that the claims of Rebholz and Northrop (1993) to have detected errors in Britton's analysis are unfounded. The three-step model used by Britton (1973) to analyse product inhibition in a mechanism with isomerization of the free enzyme is shown …