The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) 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, [ST] < Michaelis-Menten constant, KM.

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

Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST].

Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] 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 [ST] (if 116.PNGv/117.PNG[ST] is constant with coefficient of determination = 1), then KM is not applicable.