Chapter 13-14-15 Enzyme part 2 Enzyme kinetics Michaelis-Menten kinetics

Chapter 13-14-15 Enzyme part 2 Enzyme kinetics Michaelis-Menten kinetics1. Enzyme-substrate complexFormation of an enzyme-substrate complex is the first step in enzyme catalysis.中间产物学说.在酶催化的反应中，第一步是酶与底物形成酶－底物中间复合物。

当底物分子在酶作用下发生化学变化后，中间复合物再分解成产物和酶。

.许多实验事实证明了E－S复合物的存在。

E－S复合物形成的速率与酶和底物的性质有关。

.Brown(1902)和Henri(1903)提出“中间产物”假说。

(1) The existence of ES complexes has been shown in a variety of ways. ES complexes have been directly observed by electron microscopy and X-ray crystallography.. The saturation effect: at a constant concentration of an enzyme, the reaction rate increases with increasing substrate concentrations until the V max is reached.(2) Substrates are bound to a specific region of an enzyme called the active site. Much of the catalytic power of enzymes comes from their bringing substrates together in favorable orientations in enzyme-substrate (ES) complexes.. Mutations: affect the on-rate, the off-rate, kcat, …e tc. Most enzymes are highly selective in their interaction of substrates.. The active site is a three-dimensional entity formed by groups that come from different parts of the linear amino acid sequence.. 结合部位Binding site酶分子中与底物结合的部位或区域(酶的专一性). 催化部位catalytic site酶分子中促使底物发生化学变化的部位(酶催化的性质).酶的活性部位或活性中心:酶的结合部位和催化部位的总称.调控部位Regulatory site酶分子中存在着一些可以与其他分子发生某种程度的结合的部位，从而引起酶分子空间构象的变化，对酶起激活或抑制作用。

酶活性中心的通常官能团主要包括：.亲核性基团：丝氨酸的羟基；半胱氨酸的巯基；组氨酸的咪唑基。

.酸碱性基团：天冬氨酸/谷氨酸的羧基；赖氨酸的氨基；酪氨酸的酚羟基；组氨酸的咪唑基；半胱氨酸的巯基。

. The lock-and-key (锁与钥匙). The active sites of enzymes are complementary inshape to those of their substrates (Emil Fisher). The induced fit (诱导结合). the complementary active sites shapes to those of their substrates only after the substrates are bound(Daniel Koshland). Multiple weak (noncovalent) interactions between substrates and enzymes. Nonpolar character of the active sites generally. polar residues usually participate in the catalytic processes (catalytic groups with specific binding)Flexible or rigid structure. Lock-Key model of enzyme action, Emil Fischer, 1890. Induced-fit model of enzyme action, Daniel koshland, modern variation of Lock-Key model Reorientate specific functional groups into the position to catalysis (need to overcome the entropy increase)(3) Binding energy is the major source of free energy used by enzymes to lower the activation energies of reactions. Formation of each weak interaction in the ES complex is accompanied by a small release offree energy: Binding energy (ΔGb). Weak interactions are maximized when the substrate is converted to the transition state. Transition state theory. the enzyme is evolved “designed” to bind the transition state structure.2. some definition of kinetics. Velocity (速率, 反应速率) The change in the concentration of a reactant or product per unit time (单位时间内生成的产物或者底物的消失). Initial velocity (初始反应速率) The rate of the reaction immediately after mixing the enzyme and substrate. assumed that reversible reaction, linearity, …Reaction order. First-order kinetics (1级动力学). The reaction rate depends on the first order of theconcentration of a reactant.. Second-order kinetics (2级).The reaction rate depends on the concentration of tworeactants. A and B are in the rate-determining step.. Pseudo-first-order kinetics (准1级). The second-order reactions involve reactants such as water that are present in great excess. Then the reaction appears to be the first-order.. Zero-order kinetics (零级). The addition of reactant does not alter a reaction rate. The rate is constant because the reactant concentration is high enough to saturate all the catalytic sites on the enzyme molecules.3. The enzyme kineticsEnzyme kineticsThe quantitative study of enzyme catalysisprovides information about reaction rates.. Measure affinity of enzymes for substratesand inhibitors. Provide insight into catalysis or reaction mechanism. Practical application:. comprehension of the forces that regulate metabolic pathways. design of improved therapies4. Michaelis-Menton equation reflects the kinetic behavior of many enzymes. Initial velocity (Vo). measured at the beginning of the enzyme-catalyzed reaction. substrate concentration to be constant ([S] decreasedas the reaction progresses). Saturation effect (Vmax). plotting the initial velocity (Vo) against the substrate concentration ([S]). Affinity measurement (Km). Substrate concentration when velocity reached half of Vmax. Linear relationship between V o and increasing [S]. at relatively low concentrations of substrateThe investigation of enzyme rates was proposed byLeonor Michaelis and Maud Menten in 1913.单底物结合位点的单底物酶促反应Deduction of M-M equations. Quick balance assumptionE和ES之间存在平衡，ES分解生成产物的速度不足以破坏E和S之间的平衡。