Skibo E B, Gilchrist J H, Lee C H
Biochemistry. 1987 Jun 2;26(11):3032-7. doi: 10.1021/bi00385a013.
Quinazolin-4(3H)-one derivatives substituted at the 6- and/or 7-position were studied as electronic probes of substrate oxidation by buttermilk xanthine oxidase. Since the enzyme active site possesses dimensional tolerance, the substituents exert an electronic effect rather than a steric effect on the catalytic parameters for oxidation. This feature permitted a Hammett plot to be made for quinazoline-oxygen substrate activity. The concave downward nature of this plot indicates that the rate-determining step for oxidation changes when electron-withdrawing substituents are placed on the substrate. This plot and kinetic isotope effects obtained with 2-deuterio derivatives of the substrates indicate the following: (i) oxidation involves nucleophile transfer to the C(2) center in concert with hydride transfer to the molybdenum center, and (ii) the formation of oxidized product is a three-step process, i.e., Michaelis complex formation, oxidation, and hydrolysis of the oxidized substrate-enzyme adduct. The role of the nucleophile in oxidation appears to be to increase the electron density in the substrate and thereby facilitate hydride transfer. The implication of this study is that similar electronic probes may be designed to study other purine-utilizing enzymes possessing a dimensionally tolerant active site.
对在6位和/或7位被取代的喹唑啉-4(3H)-酮衍生物作为酪乳黄嘌呤氧化酶催化底物氧化的电子探针进行了研究。由于该酶的活性位点具有尺寸耐受性，取代基对氧化催化参数产生的是电子效应而非空间效应。这一特性使得能够针对喹唑啉-氧底物活性绘制哈米特图。该图向下凹的性质表明，当吸电子取代基位于底物上时，氧化的速率决定步骤会发生变化。该图以及用底物的2-氘代衍生物获得的动力学同位素效应表明如下：(i)氧化涉及亲核试剂转移至C(2)中心并同时有氢化物转移至钼中心，以及(ii)氧化产物的形成是一个三步过程，即米氏复合物的形成、氧化以及氧化底物-酶加合物的水解。亲核试剂在氧化中的作用似乎是增加底物中的电子密度，从而促进氢化物转移。本研究的意义在于，或许可以设计类似的电子探针来研究其他具有尺寸耐受性活性位点的嘌呤利用酶。
