Kinetics of enzymes with iso-mechanisms: dead-end inhibition of fumarase and carbonic anhydrase II.
Kinetics of enzymes with iso-mechanisms: dead-end inhibition of fumarase and carbonic anhydrase II.
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具有异构机制的酶的动力学：延胡索酶和碳酸酐酶 II 的死端抑制。
DOI：
10.1006/abbi.1994.1303
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发表时间：
1994
影响因子：
3.9
通讯作者：
Northrop,DB
中科院分区：
文献类型：
--
作者：
Rebholz,KL;Northrop,DB
游离酶的异构化可以在死端抑制的动力学模式中检测到，因为竞争性底物类似物在逆反应动力学中相对于产物产生非竞争性抑制。斜率和截距抑制常数的比率允许定量估计异构化对催化转化的相对动力学意义。将此动力学分析理论上应用于阴离子对牛碳酸酐酶 II 的抑制数据 [Y. Pocker 和 T. L. Deits (1982)J.是。化学。 [Sec.104, 2424] 提供了 43 ± 13% 的估计值，用于了解 pH 6.6 时异构化片段的限速情况。将实验分析应用于猪心延胡索酸酶，提供了反式乌头酸与富马酸的竞争性抑制模式，Kis= 2.0 ± 0.5 mM，以及与苹果酸的非竞争性抑制模式，Kis= 0.8 ± 0.1 mM 和 Kii= 2.3 ± 0.4 mM。假设延胡索酶的异构化片段是活性位点羧基和咪唑的再质子化，pK1= 5.53 和 pK2= 7.78 [Blanchard 和 Cleland (1980)Biochemistry19, 4506]，延胡索酸水合异构化片段的表观速率常数估计为 95 ± 22 s−1，相比之下为 42 ±化学部分为 13 s−1，完整周转为 29 ± 0.7 s−1。相比之下，苹果酸脱水的值分别为 17000 ± 5200、82 ± 25 和 82 ± 3 s−1。因此，异构化片段在富马酸水合过程中限速 30 ± 7%，但在苹果酸脱水过程中限速小于 1%。
Isomerizations of free enzyme can be detected in kinetic patterns of dead-end inhibition because competitive substrate analogs yield noncompetitive inhibition versus product in reverse reaction kinetics. The ratio of slope and intercept inhibition constants allows a quantitative estimation of the relative kinetic significance of the isomerization to a catalytic turnover. Applying this kinetic analysis theoretically to inhibition data for bovine carbonic anhydrase II by anions [Y. Pocker and T. L. Deits (1982)J. Am. Chem. Sec.104, 2424] provides an estimate of 43 ± 13% for how rate-limiting the isomerization segment is at pH 6.6. Applying the analysis experimentally to porcine heart fumarase provides a competitive pattern of inhibition bytrans-aconitate versus fumarate withKis= 2.0 ± 0.5 mM, together with a noncompetitive pattern versus malate, withKis= 0.8 ± 0.1 mM andKii= 2.3 ± 0.4 mM. Assuming that the isomerization segment of fumarase is the reprotonation of an active site carboxyl and imidazole with pK1= 5.53 and pK2= 7.78 [Blanchard and Cleland (1980)Biochemistry19, 4506], an apparent rate constant for the isomerization segment of fumarate hydration is estimated as 95 ± 22 s−1, compared to 42 ± 13 s−1for the chemical segment and 29 ± 0.7 s−1for a complete turnover. In contrast, the values are 17000 ± 5200, 82 ± 25, and 82 ± 3 s−1, respectively, for malate dehydration. Hence, the isomerization segment is 30 ± 7% rate-limiting during fumarate hydration but less than 1% during malate dehydration.