Storer A C, Carey P R
Biochemistry. 1985 Nov 19;24(24):6808-18. doi: 10.1021/bi00345a012.
The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.
将木瓜蛋白酶催化的N-苯甲酰甘氨酸和N-（β-苯丙酰基）甘氨酸的硫代甲酯水解反应的动力学常数与相应甲酯底物的动力学常数进行了比较。硫代酯的k2/Ks值比酯的k2/Ks值高2至3倍，且二者均呈现出具有相似pKa值（约为4和9）的钟形pH依赖性。硫代酯的k3值比酯的k3值小30至60倍，且不呈现pH依赖性。测定了甘氨酸两种衍生物的酯和硫代酯底物的溶剂氘同位素对k2/Ks和k3的影响。每种硫代酯底物显示出与相应酯底物相似的同位素效应。此外，质子累加技术的应用表明，与酯类情况一样，在涉及硫代酯的反应中，脱酰基过渡态中有一个质子发生转移，并且两种情况下过渡态中重原子的重组程度非常相似。发现一系列对-取代的N-苯甲酰甘氨酸酯水解反应的k3值与相应对-取代硫代酯的k3值相关[凯里，P.R.，李，H.，尾崎，Y.，&斯托勒，A.C.（1984年）《美国化学会志》106，8258 - 8262]，这表明硫醇酰基酶和二硫醇酰基酶脱酰基的速率决定步骤可能都涉及底物甘氨酸氮原子与半胱氨酸-25的硫之间接触的破坏。得出的结论是，酯和硫代酯的水解基本上通过相同的反应途径进行。由于氧-硫交换过程，N-（β-苯丙酰基）甘氨酸硫代酯底物释放的产物是二氧酸；然而，对于N-苯甲酰甘氨酸硫代酯底物，硫醇酸是初始产物。然后这种硫醇酸作为木瓜蛋白酶的底物，使酶再酰化，最终生成二氧酸产物。结果表明硫醇酸是木瓜蛋白酶的优良底物。
