Schultz R M, Varma-Nelson P, Ortiz R, Kozlowski K A, Orawski A T, Pagast P, Frankfater A
Department of Biochemistry and Biophysics, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153.
J Biol Chem. 1989 Jan 25;264(3):1497-507.
Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.
亮抑酶肽和类似的肽精氨醛（精氨酸醛）过渡态类似物蛋白酶抑制剂在水溶液中以三种共价形式存在，即亮抑酶肽水合物（IH）、通过胍基ε - N加成到醛基碳上生成的环状甲醇胺形式（IC）以及游离醛形式（IA）。D₂O中的¹H NMR显示它们的平衡浓度对于IH、IC（R和S对映体）和IA分别为42%、56%和2%。通过用氨基脲捕获IA，在D₂O中用¹H NMR测定了（化学式；见原文）的转化速率。应用2.8的氘同位素效应得出在H₂O中kC的速率常数为0.092 min⁻¹，kD的速率常数为0.73 min⁻¹。然后利用IA的平衡浓度以及kC和kD的速率来解释亮抑酶肽对组织蛋白酶B和木瓜蛋白酶抑制作用中的滞后阶段。观察到两种情况。（i）在微摩尔浓度的亮抑酶肽和木瓜蛋白酶存在下，亮抑酶肽的结合是双相的，速率常数与kD和kC相同。（ii）在总亮抑酶肽和蛋白酶更稀的纳摩尔浓度下，现在观察到的达到稳态抑制的滞后阶段（速率常数为k'）可以通过koff速率常数的低值（组织蛋白酶B为0.072 min⁻¹，木瓜蛋白酶为0.024 min⁻¹）以及活性抑制剂形式IA的极低浓度来解释，其中k' = kon[IA] + koff。虽然kon[IA]很慢，但发现二级速率常数kon相当快，组织蛋白酶B为1.2×10⁷ M⁻¹ s⁻¹，木瓜蛋白酶为1.8×10⁷ M⁻¹ s⁻¹。因此，亮抑酶肽与组织蛋白酶B和木瓜蛋白酶的结合可能会显示滞后阶段，但这并非由于结合缓慢所致。
