Influence of premixing on transient-phase kinetics for two-substrate enzyme systems

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Transient-phase kinetic equations are worked out, using the Laplace transform method, for two-substrate enzyme reactions occurring by the Theorell-Chance, ping pong bi bi and ordered ternary-complex mechanisms (the equations for the random ternary-complex mechanism are too complex to be useful). Two sets of premixing conditions were considered: (i) E not premixed with the first substrate A, i.e., E | A | B, E | A + B or E + B | A; and (ii) E premixed with the first substrate A, i.e., E + A | B. For each mechanism and premixing conditions two cases were treated: (a) enzyme concentration limiting, i.e., e 0 ⪡ a 0, b 0 , and (b) concentration of first substrate A limiting, a 0 ⪡ e 0, b 0 . In all cases (a) gives rise to a transient phase followed by a steady state, the transient phase being represented by two or more exponential terms. In all cases (b) gives no steady state; the concentrations of products X and Y rise to a final value of a 0 in a manner represented by two or more exponential terms. Premixing of type (ii) leads to a more rapid initial rise than that of type (i), in Cases a and b. Some results on horse liver alcohol dehydrogenase are shown to be consistent with the equations derived for a Theorell-Chance mechanism; there is no evidence for the participation of two types of active sites on the enzyme.