Baici A, Gyger-Marazzi M
Eur J Biochem. 1982 Dec;129(1):33-41. doi: 10.1111/j.1432-1033.1982.tb07017.x.
Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k on and k off for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio k off/k on, was in good agreement with the inhibition constant obtained using a steady-state treatment. k on was 1.8 X 10(5) M-1 s-1 and 2.0 X 10(5) M-1 s-1 for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M-1 s-1. The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 10 microM, at least. This contrasts with the predictions drawn from the value of Ki (approximately 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 microM.
亮抑蛋白酶肽被发现是一种对人脾脏和兔肝脏组织中的组织蛋白酶B具有缓慢、紧密结合特性的抑制剂。在存在抑制剂的酶催化反应过程中，在达到稳态之前会出现一个持续几分钟的浓度依赖性瞬态，根据酶是否已与抑制剂预孵育，其特征分别为向上凹或向下凹的滞后阶段。从曲线的稳态前阶段可以计算出酶-抑制剂复合物形成的结合速率常数（k on）和解离速率常数（k off）。作为k off/k on比值的抑制常数（Ki）与使用稳态处理获得的抑制常数高度一致。人源和兔源酶的k on分别为1.8×10⁵ M⁻¹ s⁻¹和2.0×10⁵ M⁻¹ s⁻¹，结合过程的缓慢符合酶滞后的一般概念。二硫苏糖醇对组织蛋白酶B必需半胱氨酸残基的激活也是一个非常缓慢的过程，其二级速率常数为4.1 M⁻¹ s⁻¹。亮抑蛋白酶肽结合的动力学特征有助于预测该抑制剂在体内对组织蛋白酶B的可能抑制效率。结果表明，为了使亮抑蛋白酶肽成为组织蛋白酶B具有生理意义的抑制剂，其在靶位点的浓度必须至少超过10 μM。这与根据Ki值（约5 nM）得出的预测结果形成对比，后者表明在浓度为0.05 μM时就已经能有效抑制该酶。
