Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex
Ikechukwu I. Udema *
Department of Chemistry and Biochemistry, Research Division, Ude International Concepts LTD (RC: 862217), B. B. Agbor, Delta State, Nigeria.
*Author to whom correspondence should be addressed.
Abstract
Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration.
Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T.
Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters.
Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST].
Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.
Keywords: Aspergillus oryzea alpha-amylase, total enzyme-substrate complex, heterogeneous mixture of undissociated enzyme-substrate complex and free enzyme, cage effect.