Lin L N, Brandts J F
Biochemistry. 1979 Jan 9;18(1):43-7. doi: 10.1021/bi00568a007.
The rates of hydrolysis of glycy-L-proline and L-phenylalanyl-L-proline, catalyzed by prolidase, have been measured at several temperatures under conditions where a high ratio of prolidase activity to substrate concentration existed. Two well-separated kinetic phases, which can be adequately treated as two first-order reactions, were observed for the hydrolysis. The relative amplitudes of the two phases are nearly independent of temperature, but strongly dependent on the initial state of protonation of the dipeptides. It was found that the amplitude of the slow phase is strictly proportional to the known amount of cis isomer, while the amplitude of the fast phase correlates with the amount of the trans isomer. Furthermore, the relaxation time and activation energy of the slow phase of hydrolysis are in good agreement with the same parameters determined for cis-trans isomerization of the dipeptides, as measured by a pH-jump method for samples not being hydrolyzed. These results lead us to the conclusion that the slow phase seen for hydrolysis is rate limited by cis-trans isomerization of the X-pro peptide bond. Thus, this proline-specific protease appears to have an absolute requirement for the trans form of the peptide bond and appears not to cleave the cis form or to cleave it extremely slowly.
在存在高脯氨酰二肽酶活性与底物浓度比的条件下，于多个温度下测定了脯氨酰二肽酶催化的甘氨酰-L-脯氨酸和L-苯丙氨酰-L-脯氨酸的水解速率。观察到水解过程有两个明显分开的动力学阶段，可充分视为两个一级反应。两个阶段的相对幅度几乎与温度无关，但强烈依赖于二肽的初始质子化状态。发现慢相的幅度与已知的顺式异构体数量严格成正比，而快相的幅度与反式异构体的数量相关。此外，水解慢相的弛豫时间和活化能与通过pH跃变法测定的未水解样品中二肽顺反异构化的相同参数非常吻合。这些结果使我们得出结论，水解中看到的慢相速率受X-脯氨酸肽键顺反异构化的限制。因此，这种脯氨酸特异性蛋白酶似乎对肽键的反式形式有绝对要求，似乎不切割顺式形式或切割极其缓慢。
