竞争性抑制的非稳态酶动力学布尔函数图论研究

以非稳戊酶动力学的布尔函数图形方法,来研究一类竞争性抑制的非稳态酶动力学问题,推导出此类反应的百稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类竞争性抑制酶反应体系的非稳态酶动力学问题。     

Ping Pong Bi Bi机制的非稳态酶动力学分布函数图论研究

以非稳态酶动力学的布尔函数图形方法,来研究一类PingPongBiBi机制的非记酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类PingPongBiBi机制酶反应体系的非稳态酶动力学方程。     

Random Bi Bi机制的非稳态酶动力学布尔函数图论研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类Random Bi Bi机制的非稳态酶动力学问题,推导同此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类Random Bi Bi机制酶反应体系的非稳态酶动力学方程。     

非稳态酶活化动力学的布尔函数图论分析

以非稳态酶动力学的布尔函数图形方法研究非稳态酶活化动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了酶活化反应体系的非稳态酶动力学过程．     

一类非竞争性抑制的非稳态酶动力学布尔函数图解研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类非竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类非竞争性抑制的非稳态酶动力学的动力学过程。     

酶动力学的极值原理假说

平衡态假说和稳态假说是酶动力学的基础。本文提出极值原理假说,用于完善平衡态假说和稳态假说的不足之处,扩大了酶动力学的应用范围,并以Michaelis-Menten方程为例进行了说明。     

酶促水解大豆分离蛋白动力学模型的研究

本文对AS1.398中性蛋白酶在pH6.9和温度49℃条件下水解大豆分离蛋白的动力学机制进行了研究．结果表明：酶水解速率随水解反呈指数递减．为了解释实验结果,我们提出了如下假设：对底物而言水解反应终为零级反应,水解过程中由于游离酶攻击酶-底物中间络合物而造成的不可逆酶变性是一个二级动力学过程．在此基础上,由实验数据推导得到了描述AS1.398中性蛋白酶催化水解大豆分离蛋白的动力学方程,该方程可用于指导和优化酶解反应实验．     

反胶束中单宁酶催化没食子酸烷基酯的生物合成

为了研究单宁酶在有机相中的催化性能,建立了AOT/异辛烷／水反胶束单宁酶催化没食子酸与脂肪醇酯合成反应体系。结果显示：反胶束单宁酶催化体系可成功催化合成C3-C5脂肪醇与没食子酸的酯合成反应。不同反应体系中由于不同脂肪醇的存在,单宁酶的动力学参数和紫外光谱存在差别。结果表明单宁酶对脂肪醇的专一性不强,根据Vmax/Km比值,丁醇与异丁醇是其最适底物,单宁酶催化没食子酸烷基酯合成的动力学符合米氏方程。反应体系中不同的脂肪醇导致了单宁酶构象的差别。     

滨海白首乌提取物对酪氨酸酶活力的影响

以l-dopa为底物,研究了白首乌提取物的酪氨酸酶活性影响作用。结果表明白首乌提取物对酪氨酸酶抑制作用显著,可使酶促反应进程出现较长迟滞期,稳态酶活力缓慢下降。白首乌提取物终浓度80.0μg/mL时,迟滞时间为626 s,稳态酶活力相对维生素C抑制率为56.25%。白首乌提取物对酪氨酸酶的抑制机理为可逆的竞争性抑制。     

双底物酶促反应动力学机制判别的优化方法

研究双底物酶促反应动力学机制的常用方法是二次作图法.本文针对二次作图法存在的问题提出了一种基于最小二乘拟合的优化算法.该算法利用最小二乘原理对顺序机制和乒乓机制下的双倒数速度方程的常系数进行拟合求解,通过比较两种拟合方程下得到的残差平方和判别酶促反应的动力学机制.通过实例计算证明该优化算法具有可靠性强、计算过程简单的特点,在双底物酶促反应动力学机制判别的准确性方面更具优势.     

Asymmetric Effect of Mechanical Stress on the Forward and Reverse Reaction Catalyzed by an Enzyme

The concept of modulating enzymatic activity by exerting a mechanical stress on the enzyme has been established in previous work. Mechanical perturbation is also a tool for probing conformational motion accompanying the enzymatic cycle. Here we report measurements of the forward and reverse kinetics of the enzyme Guanylate Kinase from yeast (Saccharomyces cerevisiae). The enzyme is held in a state of stress using the DNA spring method. The observation that mechanical stress has different effects on the forward and reverse reaction kinetics suggests that forward and reverse reactions follow different paths, on average, in the enzyme''s conformational space. Comparing the kinetics of the stressed and unstressed enzyme we also show that the maximum speed of the enzyme is comparable to the predictions of the relaxation model of enzyme action, where we use the independently determined dissipation coefficient for the enzyme''s conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.     

Transient model of thermal deactivation of enzymes

The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.     

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.     

Enzyme kinetics of muscle glycogen phosphorylase b

Interest in the kinetics of glycogen phosphorylase has recently been renewed by the hypothesis of a glycogen shunt and by the potential of altering phosphorylase to treat type II diabetes. The wealth of data from studies of this enzyme in vitro and the need for a mathematical representation for use in the study of metabolic control systems make this enzyme an ideal subject for a mathematical model. We applied a two-part approach to the analysis of the kinetics of glycogen phosphorylase b (GPb). First, a continuous state model of enzyme-ligand interactions supported the view that two phosphates and four ATP or AMP molecules can bind to the enzyme, a result that agrees with spectroscopic and crystallographic studies. Second, using minimum error estimates from continuous state model fits to published data (that agreed well with reported error), we used a discrete state model of internal molecular events to show that GPb exists in three discrete states (two of which are inactive) and that state transitions are concerted. The results also show that under certain concentrations of substrate and effector, ATP can activate the enzyme, while under other conditions, it can competetively inhibit or noncompetitively inhibit the enzyme. This result is unexpected but is consistent with spectroscopic, crystallographic, and kinetic experiments and can explain several previously unexplained phenomena regarding GPb activity in vivo and in vitro.     

Kinetics studies in a continuous steady state hollow fiber membrane enzyme reactor

Porous hollow cellulose fibers have been used to separate a nonflowing enzyme solution of alkaline phosphatase from a continuous flow of substrate. The porosity of the hollow fiber membrane allows the substrate and product to diffuse freely through the membrane while restricting the permeation of the enzyme. The resulting “immobilized” enzyme system has been shown to behave as a continuous reactor—converting p-nitrophenylphosphate to p-nitrophenol. By varying the concentrations, flow rate, etc., either diffusion or enzyme kinetics can be studied. The continual influx of product and removal of substrate at steady state allows the study of kinetics of relatively short half-life enzymes and unstable systems.