Rudnick G, Abeles R H
Biochemistry. 1975 Oct 7;14(20):4515-22. doi: 10.1021/bi00691a028.
Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.
脯氨酸消旋酶催化D - 脯氨酸和L - 脯氨酸的相互转化。本实验室先前的研究已证实，该反应通过双碱机制进行，即酶上的一个碱将底物α - 氢作为质子除去，另一个碱的共轭酸将质子给予α - 碳的另一侧（卡迪纳莱，G.J.，和阿贝莱斯，R.H.，（1968年），《生物化学》7，3970）。所提出机制的一个假设是，酶 - 底物复合物中不会发生质子交换。在本研究中，我们已表明，在脯氨酸消旋酶存在下，DL - [α - ³H]脯氨酸的³H释放速率随脯氨酸浓度的增加而降低。这些结果表明，底物衍生的质子从酶上的释放主要（可能完全）发生在产物释放之后。在初始速度条件下，L - [α - ³H]脯氨酸的³H释放速率不会随L - 脯氨酸浓度的增加而降低。因此，来自一种异构体的酶结合质子只能被另一种异构体“捕获”。我们得出结论，该酶有两种形式；一种结合L - 脯氨酸，另一种结合D - 脯氨酸。底物衍生的质子从酶上的释放比这两种形式的相互转化更快。这些结果与先前提出的机制一致。通过在十二烷基硫酸钠存在下的凝胶电泳测定，脯氨酸消旋酶由分子量为38,000的相似亚基组成。平衡透析实验检测到每两个亚基只有一个底物结合位点。当无活性且不能结合底物的酶的氧化形式被硫醇还原以产生活性酶时，每个二聚体有两个半胱氨酸巯基可用于与碘乙酸反应。当其中一个半胱氨酸被修饰时，酶会失活。所有碘乙酸的掺入都发生在酶一级序列的同一点，并且可以通过脯氨酸或吡咯 - 2 - 羧酸盐（一种底物类似物）的存在来防止。提出了一个模型，其中单个活性位点由两个相同亚基的元件形成。尽管数据与该模型一致，但另一种解释（即一半的亚基无功能）也不能排除。
