中科大基础生物化学实验实验七 酸性磷酸酯酶动力学分析

1、What does Km reveal?What is Steady State? Why do initiational velocity curve? Why do phenol Standard Curve?Kinetic Study of the Enzyme Acid Phosphatase (E.C. 3.1.3.2.)Basic Fetures of EnzymeHigh Catalytic Power: kcat /kun 107High Specificity: reaction type and substrates Proteolytic Enzymes: peptide

2、 bonds Subtilisin: any kind of peptide bond Trypsin: X-K, X-R Thrombin: blood clotting R-G in specific peptide sequence Michaelis-Menten ModelSteady StateMichaelis Constant Km: equals S at witch V0 = 1/2 Vmax If k2 100-fold)2)Improved estimation by number number of data points with S between 0.25 an

3、d 5.0 KmExperimental measurement of kcat and KmS: 250, 50, 10, 2, and 0.4 MExperimental measurement of kcat and KmS: 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 MLineweaver-Burk or double reciprocal plotExperimental measurement of kcat and KmS = 0.25 -5.0 KmLineweaver-Burk double-reciprocal plotE

4、xperimental measurement of kcat and KmEisenthal-Cornish-Browden Direct PlotsExperimental measurement of kcat and KmKm = 11.63M, Vmax = 100.36M/sExperimental measurement of kcat and KmComparasion of various graphical treatments 2.米氏常数（Km）和最大反应速度（Vm）的测定 1)酚标准曲线 Km和Vm的测定(做两组平行对照)管号12345670Ph-PO4Na2终浓度（

5、mM）0.10.20.30.50.7511.50.51/SPh-PO4Na2(ml)0.2ml (0.5mM)0.4ml (0.5mM)0.06m (5mM)0.1ml (5mM)0.15m (5mM)0.2ml (5mM)0.3ml (5mM)0.1ml (5mMBuffer（ml）0.30.10.440.40.350.30.20.4酶液（1：20）0.5ml终止后加分别在35度反应10分钟,各加2mlNa2CO3(1M）终止反应各管加0.5mlFolin乙试剂，35度显色10分钟A680相当于酚含量V1/V Km、Vm测定时间设计 1 2 3 4 5 6 0 30 1 130 2 230

6、10 1030 11 1130 12 1230 1 2 3 4 5 6 3 330 4 430 5 530 18 1830 19 1930 20 20303. NaF及KH2PO4抑制类型(可逆或不可逆)的判断-V-E曲线的制作 35度精确反应15分钟, 各管先后加入2毫升1M碳酸钠溶液和0.5毫升福林-酚稀溶液, 35度保温10分钟显色,测A680 NaF和KH2PO4可逆抑制类型（竞争性、非竞争性或反竞争性）的判断组别KH2PO4NaF管号1234567891011121314151617180（S）mM0.10.20.30.511.50.10.20.30.511.50.10.20.30.

7、511.51/sS体积（mll）0.2(0.5mM)0.4 (0.5mM0.06 (5mM0.1 (5mM0.2 (5mM0.3(5mM0.2 (0.5mM0.4 (0.5mM0.06 (5mM0.1 (5mM0.2 (5mM0.3 (5mM0.2 (0.5mM0.4 (0.5mM0.06 (5mM0.1 (5mM0.2 (5mM0.3 (5mM0.1 (5mMKH2PO40.1NaF0.1Buffer(ml)0.40.20.540.50.40.30.30.10.440.40.30.20.30.10.440.40.30.20.5酶（ml)0.40.4分别在35度反应10分钟，各管先后加入2毫升

8、1M碳酸钠溶液终止反应各管加0.5毫升福林-酚稀溶液, 35度保温10分钟显色,测A680A680数据处理用origin软件直接拟合法计算Km和Vmax.用双倒数法和EXCEL软件求算Km和Vmax.用Eisenthal-Cornish-Browden直接法求算Km和Vmax.用origin软件和双倒数法分别求算抑制剂的Ki值。如果酶的浓度为1uM，计算kcat。实验中应注意的问题： 1. 所有实验中空白管中酶最后加（即在加过底物、 NaCO3、Folin-酚试剂后加入） 2. 各溶液在试管中必需混匀.实验中应注意的问题3酚标准曲线实验，ddH2O改为0.2M、PH5.6乙酸盐buffer。4实验二中Km、Vm测定做两组。实验三只需做一组。 Km、Vm测定时间设计： 1 2 3 4 5 6 0