Nys P S, Savitskaia E M, Klesov A A, Sinitsyn A P, Shviadas V Iu
Antibiotiki. 1978 Jan;23(1):46-50.
The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.
研究了青霉素酰胺酶在40℃温度下失活速率常数的pH依赖性。结果表明，在所有情况下，酶失活均符合一级反应动力学。发现pH依赖性曲线呈钟形，失活速率常数从最高值转变为最低值时，其变化超过100倍。根据所获得的数据以及先前发表的数据，得出结论：酶失活按照如下机制进行，即青霉素酰胺酶的三种平衡离子形式，即“酸性”、“中性”和“碱性”中，活性酶的中性形式最稳定。对该机制进行了动力学分析，结果表明所发现的依赖性与理论曲线相符，在40℃温度下，控制青霉素酰胺酶形式间相互转化的离子基团的pK值分别为2.4和10.1。“酸性”或“碱性”形式的失活速率常数为5.95 min⁻¹，而酶的“中性”形式的失活速率常数为5.1×10⁻⁴ min⁻¹。提出了酶失活的机制。根据该机制，青霉素酰胺酶天然结构中盐桥的破坏导致产生了与天然形式相比极其不稳定的酶形式。
