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.

这项研究描述了基于新的积分方程的新方法的开发，该积分方程可以确定两种互不排斥的抑制剂的动力学参数，其中一种抑制剂是在时程反应中产生的。同时被磷酸盐和尿素抑制的碱性磷酸酶被用来说明这种方法，包括评估它们之间的相互作用。使用两个积分速度方程式进行数据分析：排他性线性混合抑制（EMI）和非排他性线性混合抑制（NEMI）。使用非线性回归估计动力学参数，结果表明：（i）酶与抑制剂脲和磷酸盐之间的相互作用表现出互不排斥的行为；（ii）更具体地说，这些抑制剂仅在游离酶（E）种类中是非排他性的；（iii）抑制剂也显示出与被归类为促进作用的酶的相互作用；（iv）磷酸盐是竞争性抑制剂，尿素是混合抑制剂；（v）磷酸盐的抑制常数远低于尿素的测定常数。此外，还包括可适用于任何动力学研究的功能性Excel电子表格作为补充。