Enzymatic_reaction_kinetics.ppt

Section 5 Enzymatic Reaction Kinetics5.1 The effects of substrate concentration on reaction rateSubstrate saturation curve for an enzyme-catalyzedReaction. The amount of enzyme is constant, and theVelocity of the reaction is determined at variousSubstrate concentration.* At low concentration, the reaction rate is increased as the increase of substrate concentration. dp/dt = k [S]* At relative high concentration, the reaction rate is increased as the increase of substrate concentration, but the increase rate is not in direct proportion* At high concentration, the reaction rate is not increased as the increase of substrate concentration. dp/dt = k [Et] Where Et is the total concentration of enzyme The relationship of reaction rate and substrate concentration can be explained by intermediate theory. E + S = ES → E + P The maximal rate means all the enzyme are saturated by substrates. k1 k3E + S = ES → E + P k2The enzyme (E), combines with its substrate (S) to form an enzyme-substrate complex (ES). The (ES) complex can dissociate again to form E + S, or can proceed chemically to form E and the product P. The michaelis-Menten equation:1913Michaelis-menten constant Km (米氏常数米氏常数)Vmax / 2 = Vmax . [S] / Km + [S] Km = [S]Km is equivalent to the substrate concentration at which the velocity is equal to half of Vmax . 米氏常数是酶促反应速度为最大米氏常数是酶促反应速度为最大反应速度一半时的底物浓度。

反应速度一半时的底物浓度 Significances of Km A measure of the affinity (亲和力亲和力) of an enzyme for its substrate Km is a measure of the stability of the ES complex, being equal to the sum of the rates of breakdown of ES over its rate of formation. For many enzymes k2 is much great than k3. The value of Km depends on the relative values of k1 and k2 for formation and dissociation,respectively.Km=(k2 + k3)/ k1vA high Km indicates weak substrate binding ( k2 predominant over k1 ), a low Km indicates strong substrate binding (k1 predominant over k2 )问题：问题：如果已知一种酶有几种底物，如何确定最佳底如果已知一种酶有几种底物，如何确定最佳底物？物？vKm is the specific to a given enzyme. It is the featuring constant of enzyme. At given condition and substrate, an enzyme has its specific Km to the substrate. Measurement of KmvBecause Vmax is achieved at infinite substrate concentration, it is impossible to estimate Vmax (and hence Km) from a hyperbolic plot 双双曲线曲线. Vmax and Km can be determined experimentally by measuring v at different substrate concentrations.（（a a））估计估计V V和和K Km m，（，（b b））在在[S]=2.5×10[S]=2.5×10-5-5molLmolL-1-1和和[S]=5.0×10[S]=5.0×10-5-5molLmolL-1-1时，时，v v会是多大？会是多大？Lineweaver-burk plot (double reciprocal plot)-1/Km1/Vmaxslope=Km/VmaxHanes-Wolff plot5.2 Bi-substrate and Bi-product Reaction A+BP+Q sequential reactions（序列反应（序列反应 ）） ping pong reactions（乒乓反应（乒乓反应 ）） orderd reactions（有序反应（有序反应 ）） random reactions（随机反应（随机反应 ）） 5.2.1 ordered reactions（序列有序反应（序列有序反应）） 5.2.2 random reactions（序列随机反应（序列随机反应）） 5.3.3 ping pong reactions（乒乓反应（乒乓反应）） 5.3 The effects of enzyme concentration on reaction rate[S]>>[E]V∝[E] 5.4 pH values• each enzyme has an optimum pH at which the rate of the reaction that it catalyzes is at its maximum.• small deviations in pH from the optimum value lead to decreased activity due to change in the ionization of groups at the active site of the enzyme.• large deviations in pH lead to denaturation of enzyme protein itself, due to interference with the many weak noncovalent bonds maintaining its three-dimensional structure.A graph of v plotted against pH will usually give a bell-shaped curve:The pH activity profile of four different enzyme.vMany enzyme have a optimum pH （最适）（最适）of around 6.8, but there is great diversity in pH optima of enzymes, due to the different environments in which they are adapted to work. For example, the digestive enzyme pepsin 胃蛋白酶胃蛋白酶 is adapted to work at the acidic pH of the stomach (around pH 2.0) 酶的最适酶的最适pH不是一个固定的常数不是一个固定的常数，它受到底，它受到底物的种类、浓度物的种类、浓度; 缓冲液的种类、浓度缓冲液的种类、浓度; 酶的纯酶的纯度度;反应的温度、时间等的影响。

反应的温度、时间等的影响例例: 碱性磷酸酶催化磷酸苯酯水解时碱性磷酸酶催化磷酸苯酯水解时 [s]为为2.5x10-5 M，，最适最适pH为为 8.3 [s]为为2.5x10-2 M，，最适最适pH为为10.0 5.5 TemperatureTemperature affects the enzyme-catalyzed reaction in two ways:(1) A rise in temperature increases the thermal energy 热能热能of the substrate molecules. This raises the proportion 比例比例of substrate molecule with sufficient energy to overcome the energy barrier能障能障 , and hence increase the rate of the reaction.v(2) A second effect comes into play at higher temperature. Increasing the thermal energy of the molecules which make up the protein structure of the enzyme itself will increase the chances of making the multiple weak non-covalent interactions which hold the three-dimensional structure of the enzyme together.The overall effect of a rise in temperature on the reaction rate of enzyme is a balance between these two opposing effects. A graph of temperature plotted against velocity v will therefore show a curve, with a well defined temperature optimum.For many mammalian enzymes this is around 37℃℃, but there are also organisms which have enzymes adapted to working at considerably higher or lower temperatures. For example, Taq polymerase 聚合酶聚合酶, which is used in the polymerase chain reaction （（PCR 聚合酶链式反应聚合酶链式反应 ））is found in a bacterium that lives at high temperatures in hot springs（（温泉）温泉）, and thus is adapted to work optimally （（最适最适合的）合的）at high temperatures.5.6 ActivatorsAny molecule which can raise the activity of an enzyme is called activator of the enzyme. many activator are metal ions or minerals. Mg2+, Mn2+, Co2+, Cl- are common activators for many enzymes. Activator is a type of co-factor 辅因子辅因子 with loose combination to enzyme. They are very easy to be washed out during the proceeding of enzyme purification. To recover the active of a purified enzyme, activators are added to the reaction system. 5.7 Inhibitors•Any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor. •Some enzyme inhibitors are normal cellular metabolites 代谢物代谢物that inhibit a particular enzyme as part of the normal metabolic control of a pathway 途径途径.•Other inhibitors may be foreign substances, such as drugs 药物药物or toxins毒物毒物, where the effect of enzyme inhibition could be either therapeutic 治疗性的治疗性的or, at the other extreme 另另一个极端一个极端, lethal 致命的致命的. Enzyme inhibition may be of two main types:Irreversible 不可逆不可逆or reversible可逆可逆, which reversible inhibition itself being subdivided into competitive 竞争性竞争性and noncompetitive 非竞争性非竞争性 inhibition and uncompetitive反竞争性反竞争性inhibition. Reversible inhibition can be overcome by removing the inhibitor from the enzyme, for example by dialysis, but this is not possible for irreversible inhibition, by definition.Irreversible inhibitionvInhibitors which bind irreversibly to an enzyme often form a covalent bond to an amino acid residue at or near the active site, and permanently inactivate （（永久性失活）永久性失活）the enzyme. vSusceptible （（有关联的）有关联的）amino acid residues include Ser and Cys residues which have reactive –OH and –SH groups, respectively.vDiisopropylphosphofluoridate (DIPF二异丙基氟磷酸二异丙基氟磷酸 ), a component of nerve gases（（神经毒气）神经毒气）, reacts with a Ser residue in active site of the enzyme acetylcholinesterase （（乙酰胆碱酯酶）乙酰胆碱酯酶）, irreversibly inhibiting the enzyme and preventing the transmission of nerve impulses.vIodoacetamide (碘乙酸碘乙酸)modifies (修饰修饰) Cys residues and hence may be used as a diagnostic tool in determining whether one or more Cys residues are required for enzyme activity.vThe antibiotic penicillin irreversibly inhibits the glycopeptide transpeptidase （（糖肽转肽酶）糖肽转肽酶）enzyme that forms the cross-links in the bacterial cell wall by covalently attaching to a Ser residue in the active site of the enzyme.Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidasewhich catalyzes an step inbacterial cell wall synthesispenicillin consists of athiazolidine ring fused toβ-lactam ring to which avariable group R is attacheda reactive peptide bondin the β-lactam ring covalently attaches to aserine residue in the activesite of glycopeptide tran-sition.Identification of inhibitionReversible inhibition1 Competitive inhibitionvA competitive inhibitor typically has close structural similarities to the normal substrate for the enzyme. Thus it competes with substrate molecules to bind to the active site.vThe enzyme may bind either a substrate molecule or an inhibitor molecule, but not both at the same time.vThe competitive inhibitor binds reversibly to the active site. At high substrate concentrations the action of a competitive inhibitor is overcome because a sufficiently high substrate concentration will successfully compete out the inhibitor molecule in binding to the active site. Thus there is no change in the Vmax of the enzyme but the apparent affinity of the enzyme for its substrate decreases in the presence of the competitive inhibitor, and hence increases. vA good example of competitive inhibition is provided by succinate dehydrogenase 琥珀酸脱氢酶琥珀酸脱氢酶. This enzyme uses succinate琥珀酸琥珀酸as its substrate and is competitively inhibited by malonate 丙二酸丙二酸 which differs from succinate in have one rather than two methylene groups 亚甲亚甲基基.Structure of succinate , the substrate succinate dehydrogenase and malonate, the competitive inhibitor.E + S ES E + P + I EIv = V [S] / Km (1 + [I] / Ki) + [S]Lineweaver-Burk plot of competitive inhibition Lineweaver-Burk plotFinal results: 1. Unchanged of velocity v =Vmax,2. Increased of Km Noncompetitive inhibitionvA noncompetitive inhibitor binds reversibly at a site other than the active site and causes a change in the overall three-dimensional shape of the enzyme that leads to a decrease in catalytic activity.vSince the inhibitor binds at a different site to the substrate, the enzyme may bind the inhibitor, the substrate or both the inhibitor and substrate together.vThe effects of a noncompetitive inhibitor cannot be overcome by increasing the substrate concentration.vIn noncompetitive inhibition the maximum velocity Vmax is decreased. vIn noncompetitive inhibition the affinity of the enzyme for the substrate is unchanged and so Km remains the same.E + S ES E + P + + I I EI + S ESI v = V /(1 + [I] / Ki) [S] / Km + [S]Lineweaver-Burk plot of noncompetitive inhibition Final results: 1. Unchange of Km 2. Change of velocityuncompetitive inhibition （（ 反竞争性抑制作用）反竞争性抑制作用） SummaryApplications of enzyme inhibitionvBe used to investigate the activity site of a enzymevBe used to design the medicine or pesticides (杀虫剂杀虫剂)vVmax? Km? Type of inhibition?[S](μmol/L)V（（μmol/L））/min无抑制剂无抑制剂有抑制剂有抑制剂3510309010.414.522.533.840.54.16.411.522.633.8Section 6 Allosteric enzyme（（别构酶）别构酶）vSome enzymes have the allosteric feature similar to hemoglobin. These enzymes are called allosteric enzymes.vThe properties of Allosteric enzymes1.They are oligomers with higher molecular weight. Besides the substrate binding site at active region, there is regulator binding site at regulatory region. Both regions may be in either different subunits or the same subunit. 2. There is allosteric effect别构效应别构效应 allosteric activator for positive effect allosteric inhibitor for negative effect 3. A plot of v against [s] gives a sigmoidal curve (S 形曲线形曲线)rather than the hyperbolic plots (双曲线双曲线) predicted by the Michaelis-Menten equation. Their v versus [S] plotw yield sigmoid or S-shaped curve rather than rectangular hyperbolasHeterotropic allosteric effects. A and I binding to R andT,respectively.Glycogen phosphorylaseSection 7 Determination of enzyme activity 酶活性测定酶活性测定vDetermination: an enzyme activity can be determined by measuring the rate of appearance of product or the rate of disappearance of substrate.vThe change of product and substrate may be measured by following the change of absorbance at a specific wavelength using a spectrophotometer （（分光光度计）分光光度计）.vTwo of the most common molecules used for absorbance measurement in enzyme assays are the coenzymes NADH and NADPH which each absorb in the ultraviolet region at 340nm.CH3CH(OH)COO- + NAD+ CH3COCOO- + NADH+H+ Lactate pyruvatevEnzyme unitsvEnzyme activity may be expressed in a number of ways. The most commonest is by the initial rate of the reaction being catalyzed: μmol of substrate transform per minute; μmol /min or μmol min-1.vEnzyme unit (U): amount of enzyme which will catalyze the transformation of 1 μmol of substrate per minute t 25 ℃℃ under optimal conditions for that enzyme.vKatal (kat): catalytic activity which will raise the rate of the reaction by one mole per second in a specified system. 1 kat = 6×107U1U = 16.67 nanokatvSpecific activity比活力比活力: the term activity refers to the total units of enzyme in sample, whereas the specific activity is the number of enzyme units per milligram of protein (unit/mg). So the specific activity is a measure of the purity of an enzyme.。