The life span of steps in the enzyme-catalyzed reaction, its implications, and matters of general interest

There is increasing recognition for steps and their life span (LS) in the enzyme-catalyzed reaction pathway; the need for rate constants, activation parameters, and transition states (TS) has assumed preeminence in the literature. The determination of the life span of complexes, enzyme-product (EP) complexes in particular, is not a regular feature in most studies. The study aims to show that there is LS for EP and TS destined for irreversible product formation and release. The cognate objectives are to: 1) derive the equation for the graphical determination of the first-order rate constant (FORC) of EP dissociation into E and P as well as the FORC of ES dissociation to free enzyme, E, and free substrate, S; 2) derive a FORC equation for the TS to EP conversion; 3) calculate the duration; and 4) calculate the FORC for the TS to EP conversion. Using mesophilic alpha-amylase and the Bernfeld method of assay, the velocities of catalysis were used to generate first the Michaelian parameters and then the rate constants. The LS of the TS and the FORC for the conversion of the enzyme-substrate (ES) complex to TS are 4.321 exp. (-6) min and 2.314 exp. (+5)/min, respectively; the LS of the TS destined for backward conversion (deactivation) to ES and its FORC are 7.525 exp. (-7) min and 1.387 exp. (+6)/min, respectively; the LS of the EP and its FORC for dissociation to E and P are 1.33 exp. (-5) and 7.52 exp. (+4)/min respectively. In conclusion, there are always steps in an enzyme-catalyzed reaction in the catalytic cycle; the LS of each step is shorter than the total LS of deactivation and dissociation processes; this is applicable to forward reactions. Thermodynamic activation parameters must account for all FORC in accordance with the additivity principle.