Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas, CITAB, Universidade de Trás-os-Montes e Alto Douro, UTAD, 5000-801, Vila Real, Portugal.
Appl Biochem Biotechnol. 2020 Jun;191(2):657-665. doi: 10.1007/s12010-019-03213-9. Epub 2019 Dec 16.
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.
本研究描述了一种新方法的发展，该方法基于一个新的综合方程，可以确定当其中一种抑制剂在时程反应中产生时，两种相互非排他性抑制剂的动力学参数。同时受磷酸盐和尿素抑制的碱性磷酸酶被用来举例说明这种方法，包括它们之间相互作用效应的评估。数据分析使用两个综合速度方程：排他性线性混合抑制（EMI）和非排他性线性混合抑制（NEMI）进行。使用非线性回归估计动力学参数，结果表明：（i）酶与抑制剂尿素和磷酸盐之间的相互作用表现出相互非排他性；（ii）更具体地说，这些抑制剂仅在游离酶（E）物种中是非排他性的；（iii）抑制剂还表现出与酶的相互作用，被归类为促进；（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抑制常数。此外，还包括一个可适应任何动力学研究的功能 Excel 电子表格作为补充。
