Rebholz K L, Northrop D B
Division of Pharmaceutical Biochemistry, School of Pharmacy, University of Wisconsin, Madison 53706.
Arch Biochem Biophys. 1994 Jul;312(1):227-33. doi: 10.1006/abbi.1994.1303.
Isomerization 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. Soc. 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 by trans-aconitate versus fumarate with Ki(s) = 2.0 +/- 0.5 mM, together with a non-competitive pattern versus malate, with Ki(s) = 0.8 +/- 0.1 mM and Kii = 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) Biochemistry 19, 4506], an apparent rate constant for the isomerization segment of fumarate hydration is estimated as 95 +/- 22 s-1, compared to 42 +/- 13 s-1 for the chemical segment and 29 +/- 0.7 s-1 for 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.
在终产物抑制的动力学模式中可以检测到游离酶的异构化，因为竞争性底物类似物在逆反应动力学中对产物产生非竞争性抑制。斜率和截距抑制常数的比值可以定量估计异构化对催化周转的相对动力学意义。将这种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年）《美国化学会志》104, 2424]，可以估计在pH 6.6时异构化段的限速程度为43±13%。将该分析实验应用于猪心富马酸酶，反式乌头酸对富马酸的抑制呈现竞争性模式，Ki(s)=2.0±0.5 mM，对苹果酸则呈现非竞争性模式，Ki(s)=0.8±0.1 mM且Kii = 2.3±0.4 mM。假设富马酸酶的异构化段是活性位点羧基和咪唑的再质子化，pK1 = 5.53且pK2 = 7.78 [Blanchard和Cleland（1980年）《生物化学》19, 4506]，富马酸水合异构化段的表观速率常数估计为95±22 s-1，相比之下，化学段为42±13 s-1，完整周转为29±0.7 s-1。相比之下，苹果酸脱水时的值分别为17000±5200、82±二十5和82±3 s-1。因此，在富马酸水合过程中，异构化段的限速程度为30±7%，而在苹果酸脱水过程中则小于1%。
