Asian Journal of Research in Biochemistry

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([ES_T]) which accounts for sequestration.

Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [S_T] <  Michaelis-Menten constant, K_M.

Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [ES_T] and the enzyme-substrate complex ([ES]) prepared for product, P, formation.

Results: The [ES_T] is < [E]; [ES_T] and pseudo-first order constant, k decreased with increasing [S_T] and increased with increasing concentration of enzyme [E_T] while velocity amylolysis, v and maximum velocity of amylolysis, v_max expectedly increased with increasing [E_T] and [S_T].

Conclusion: The fact is that the [ES_T] is lower than what is usually referred to as free enzyme ([E_T] - [ES]). Therefore, if the additional part of [ES_T] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [K_M] > [S_T] is that v a 1/[E], v a [E][S_T] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [S_T] (if v/[S_T] is constant with coefficient of determination = 1), then K_M is not applicable.