A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study.

This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.