Dodgson S J, Gros G, Krawiec J A, Lin L, Bitterman N, Forster R E
Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6085.
J Appl Physiol (1985). 1990 Jun;68(6):2443-50. doi: 10.1152/jappl.1990.68.6.2443.
The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.
在25℃和pH 7.4条件下，采用三种不同技术测定了牛碳酸酐酶（BCA）催化的二氧化碳（0.002M）水合速度：两种初始速率（稳态）停流法，一种使用玻璃pH电极（在汉诺威，方法1），另一种使用pH指示剂的分光光度测量法（在费城，方法2），以及一种交换法，其中在平衡状态下测定碳酸氢盐溶液中C18O16O的消失情况（在费城，方法3）。方法1、2和3的米氏常数（Km）以及氯离子（Ki,Cl）和乙氧唑胺（Ki,ez）的抑制常数相同。方法1、2和3的周转数分别为270,000、400,000和555,000 s-1。方法2和3同时对同一BCA溶液测得的二氧化碳水合速度值相同。通过方法2和3在37℃和pH 7.2条件下从正常人溶血产物中获得的Km、最大反应速度（Vmax）、Ki,ez和Ki,Cl相同。方法1和3对兔比目鱼肌匀浆中碳酸酐酶同工酶CA III的Km和Vmax也相同。根据米氏理论，方法3获得的Km和Vmax值应明显小于方法1和2获得的值。我们得出结论，在生理条件下催化步骤本身显然不是限速步骤，并且方法3可用于获得碳酸酐酶的米氏特征。
