Gallo K A, Tanner M E, Knowles J R
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Biochemistry. 1993 Apr 20;32(15):3991-7. doi: 10.1021/bi00066a020.
The mechanism of the cofactor-independent glutamate racemase from Lactobacillus has been studied. The possible formation of an acylenzyme intermediate during catalysis has been investigated using 18O-carboxyl labeled glutamate. The absence of any washout of label during racemization argues against intermediate formation. The observation of the enzyme-catalyzed incorporation of deuterium at the C-2 position of glutamate upon racemization in D2O provides evidence for a deprotonation/protonation mechanism. Further experiments have been performed in order to determine the number of enzymic bases responsible for racemization. Solvent deuterium is efficiently incorporated into the product enantiomer but not into the recovered substrate enantiomer in each reaction direction. This finding is consistent with a "two-base" mechanism in which one enzymic base deprotonates the substrate, and the conjugate acid of a second enzymic base protonates the resulting intermediate from the opposite face. It also suggests that the two bases are monoprotic. The possibility that the two enzymic forms, which differ at the very least by the protonation states of the active-site bases, are kinetically significant has been examined by measuring the entire time course of the approach to equilibrium at various concentrations of glutamate. An "oversaturated" regime [Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529-2537] was not observed using glutamate concentrations as high as 100 mM, indicating that the two enzyme forms are rapidly interconverting under physiological conditions.
对来自乳酸杆菌的不依赖辅因子的谷氨酸消旋酶的机制进行了研究。使用18O-羧基标记的谷氨酸研究了催化过程中酰基酶中间体的可能形成。消旋化过程中没有任何标记物的洗脱，这与中间体的形成相悖。在D2O中消旋化时，观察到酶催化氘在谷氨酸C-2位的掺入，这为去质子化/质子化机制提供了证据。为了确定负责消旋化的酶碱数量，还进行了进一步的实验。在每个反应方向上，溶剂氘有效地掺入产物对映体中，但不掺入回收的底物对映体中。这一发现与“双碱”机制一致，即一个酶碱使底物去质子化，第二个酶碱的共轭酸从相反的面使生成的中间体质子化。这也表明这两个碱是单质子的。通过测量在各种谷氨酸浓度下达到平衡的整个时间过程，研究了至少在活性位点碱的质子化状态上有所不同的两种酶形式在动力学上是否重要。使用高达100 mM的谷氨酸浓度未观察到“过饱和”状态[Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529 - 2537]，这表明这两种酶形式在生理条件下迅速相互转化。
