Moore G J, Benoiton N L
Can J Biochem. 1975 Jul;53(7):747-57. doi: 10.1139/o75-102.
The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers beta-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration. Examination of Lineweaver-Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and kcat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme-substrate complex to give products. Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neurtal dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme-substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier. The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.
在修饰剂β-苯丙酸、环己醇、苄氧羰基甘氨酸（Bz-Gly）和苄氧羰基甘氨酰甘氨酸（Bz-Gly-Gly）存在的情况下，羧肽酶B（CPB）对苄氧羰基甘氨酰赖氨酸（Bz-Gly-Lys）和苄氧羰基甘氨酰苯丙氨酸（Bz-Gly-Phe）的初始水解速率会增加。三肽苄氧羰基甘氨酰甘氨酰苯丙氨酸（Bz-Gly-Gly-Phe）的水解也会被Bz-Gly和Bz-Gly-Gly激活，但这些修饰剂均不会激活CPB对苄氧羰基甘氨酰甘氨酰赖氨酸（Bz-Gly-Gly-Lys）、Z-亮氨酰丙氨酸苯丙氨酸（Z-Leu-Ala-Phe）或苄氧羰基甘氨酰苯乳酸的水解。除环己醇外，所有修饰剂在高浓度时均表现出抑制性结合模式。在固定浓度的Bz-Gly存在下对Lineweaver-Burk图的研究表明，CPB对中性和碱性肽水解的激活作用，如在外推参数Km(app)和kcat值中所反映的，是通过不同机制发生的。对于Bz-Gly-Gly-Phe，激活是因为酶-修饰剂复合物对底物的亲和力高于游离酶，而苄氧羰基甘氨酰赖氨酸水解的激活则源于酶-底物复合物分解为产物的速率增加。环己醇与Bz-Gly和Bz-Gly-Gly不同，它与所检测的任何底物均无抑制性结合模式，仅激活CPB对二肽的水解，且对碱性二肽水解的影响比对中性二肽的影响更大。此外，当以苄氧羰基甘氨酰赖氨酸为底物时，环己醇通过增加酶-底物结合亲和力和催化步骤的速率来激活CPB对其的水解，这一效应与以Bz-Gly为修饰剂时观察到的不同。CPB的异常动力学行为与羧肽酶A非常相似，这充分表明这两种酶在各自活性位点及其周围具有非常相似的结构。有人提出在CPB活性位点裂隙下方存在一个激活剂分子结合位点，以解释所观察到的动力学行为。
