Riddles P W, de Jersey J, Zerner B
Proc Natl Acad Sci U S A. 1978 Jan;75(1):172-4. doi: 10.1073/pnas.75.1.172.
A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [(14)C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 1/10 the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [(14)C]acetylchymotrypsin and by the formation of [(14)C]acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with K(cat) for the turnover of p-nitrophenyl acetate by alpha-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et al. has been shown to be due to the presence of 10% (vol/vol) isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by alpha-chymotrypsin and negate the existence of the proposed additional acetyl-enzyme intermediate.
最近一篇论文[奇伯，B.A.K.，托米奇，J.M.，默茨，E.T.和维斯瓦纳塔，T.（1977年）《美国国家科学院院刊》74卷，510 - 514页]提出了一些证据，这些证据被认为支持乙酰胰凝乳蛋白酶脱乙酰化过程中存在中间体。据观察，以跟踪[(14)C]乙酰胰凝乳蛋白酶的减少（酸沉淀放射性的减少）来衡量的脱酰基作用，其发生速率是再活化速率的1/10，再活化速率是以对N - 乙酰 - L - 酪氨酸乙酯的活性恢复来衡量的。我们的实验表明，在pH 6时，脱酰基速率常数（通过[(14)C]乙酰胰凝乳蛋白酶的损失和[(14)C]乙酸盐的形成来测量）与再活化速率常数（通过测定再活化酶对N - 乙酰 - L - 色氨酸乙酯的活性来测量）以及α - 胰凝乳蛋白酶催化对硝基苯乙酸周转的K(cat)相同（在实验误差范围内）。奇伯等人观察到的再活化速率快10倍的部分原因已被证明是他们的再活化混合物中存在10%（体积/体积）的异丙醇，并且有人认为这种效应的其余部分是整个再活化实验中同时存在10 mM N - 乙酰 - L - 酪氨酸乙酯所产生的“吲哚效应”的体现。所呈现的结果与α - 胰凝乳蛋白酶的三步催化机制完全一致，并否定了所提出的额外乙酰化酶中间体的存在。
