BACKGROUND Search for new drug targets is becoming imperative these days, given that marketed chemotherapeutic drugs have lost their efficacy against harmful agents because of adaptability to climatic changes and co-evolving vectors to new hosts. In the wake of such a challenge, the prominence of biochemical studies is increasing by way of exploring selective enzymes and investigating their structural and functional properties through biochemical kinetic parameter Km for the application of IC50 using designed drugs. Recently, discovered Adenine Aminohydrolase (EC 3.5.4.2) in Leishmania has been found to be absent in mammalian purine salvage pathway and thus considered as a promising drug target against infectious agents. OBJECTIVES The objective of this study is to isolate and characterize AAH by learning its kinetic mode of action using preferred substrate Adenine and additives estimated through expected product formation Hypoxanthine. Bioassays designed to measure exact Enzyme kinetic parameter Km value through establishing hyperbolic curve of an enzyme reaction with the use of exact values of cellular quantities for IC50 application under experimental conditions devised by presteady state approach for SSA validity. METHODS Following saturation kinetic, the plot of hyperbolic equilibrium curve developed using initial rates of product formation as a function of (Si) through forward shift under circumstance dG0 the system allows product and reactant favored reactions in relation to (Ef) ≈ [E0 = KM] until complete saturation and estimates Km and Vmax of enzyme system under applied conditions. M-M equation used to assess experimental initial rate data for estimation of Km on excel using Solver and nonlinear least square coefficient correlation "R2" using logarithmic equation for nonlinear curve assessment. RESULTS UV/Vis spectrophotometer selectively analyzed reacting components confirming Enzyme characteristic reaction constant Km equal toi15. 0 ± 2 μ mol acquired from the Hyperbolic curve developed through the use of exact (Si) ranges at selected parameter Km and Vmax. The curve assessed by Michaelis Menten equation provides Km value=14.99 μmol and non-linear least square coefficient correlation "R2" value equal to 0.9895, along with that optimized lysis buffer formulation. In the docked complexes, the interactive amino acids identified were MSE441, ALA 364, GLN363, MSE518, VAL362, GLY517, ASP538, ALA445, TYR521, and TYR444. 2D interactions revealed hydrophobic and alkyl interactions at the noncompetitive binding site of the enzyme and therefore recommended as potential inhibitors against 3ICS protein. CONCLUSION This study encourages biochemical analysis of the novel enzymes with the use of presteady state rationale in association with the computational tools as an effective way of designing drugs in a short time against selective enzymes to meet the current challenge efficiently.

中文翻译：

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利什曼原虫腺嘌呤氨基水解酶作为药物靶点的分离和表征。

背景技术由于对气候变化的适应性和与新宿主共同进化的载体，已上市的化学治疗药物已经失去了对抗有害药物的功效，因此寻找新的药物靶标变得势在必行。在这样的挑战之后，通过探索选择性酶并通过生化动力学参数 Km 研究其结构和功能特性，以使用设计的药物应用 IC50，生化研究的重要性正在增加。最近，在利什曼原虫中发现的腺嘌呤氨基水解酶 (EC 3.5.4.2) 被发现在哺乳动物嘌呤补救途径中不存在，因此被认为是一种有希望的针对感染因子的药物靶标。目的 本研究的目的是通过使用首选底物腺嘌呤和通过预期产物形成次黄嘌呤估计的添加剂来了解 AAH 的动力学作用模式，从而分离和表征 AAH。生物测定旨在通过建立酶反应的双曲线曲线来测量精确的酶动力学参数 Km 值，使用细胞数量的精确值在 SSA 有效性的预稳态方法设计的实验条件下应用 IC50。方法 在饱和动力学之后，使用初始产物形成速率作为 (Si) 的函数绘制的双曲线平衡曲线图，在 dG0 情况下，系统允许与 (Ef) ≈ [E0 = KM] 相关的产物和反应物有利于反应，直到完全饱和和估计应用条件下酶系统的 Km 和 Vmax。MM 方程用于评估实验初始速率数据，用于使用 Solver 在 Excel 上估计 Km，非线性最小二乘系数相关性“R2”使用对数方程进行非线性曲线评估。结果 UV/Vis 分光光度计选择性地分析反应组分，确认酶特征反应常数 Km 等于 i15。通过在选定参数 Km 和 Vmax 下使用精确 (Si) 范围开发的双曲线获得 0 ± 2 μ mol。Michaelis Menten 方程评估的曲线提供了 Km 值 = 14.99 μmol 和等于 0.9895 的非线性最小二乘系数相关“R2”值，以及优化的裂解缓冲液配方。在对接复合物中，鉴定的相互作用氨基酸是 MSE441、ALA 364、GLN363、MSE518、VAL362、GLY517、ASP538、ALA445、TYR521 和 TYR444。2D 相互作用揭示了酶的非竞争性结合位点的疏水性和烷基相互作用，因此推荐作为 3ICS 蛋白的潜在抑制剂。结论 本研究鼓励使用与计算工具相关的前稳态基本原理对新型酶进行生化分析，作为在短时间内针对选择性酶设计药物以有效应对当前挑战的有效方法。