Ren S, Sakai K, Schwartz L B
Department of Internal Medicine, Virginia Commonwealth University, Richmond 23298, USA.
J Immunol. 1998 May 1;160(9):4561-9.
At neutral pH, human mast cell beta-tryptase is stabilized in its enzymatically active, tetrameric form by heparin, and resists inhibition by biologic protease inhibitors. After dissociation of beta-tryptase from heparin, active tetramers rapidly convert to inactive monomers in an isotonic, neutral pH environment. Although reversible transition states probably exist during this conversion, once inactive monomers form, addition of heparin fails to reconstitute active tetramer at neutral pH. The current study shows that complete reactivation of inactive monomers can occur at acidic pH in a heparin-independent manner. The respective rate-determining steps for formation of tetramer and active enzyme from inactive monomers exhibit second and first order kinetics based on an analysis of initial reaction rates. The optimal pH for tetramer formation and reactivation is about 6, suggesting His residues play a critical role. The optimal ionic strength equivalent is 160 mM NaCl; and the optimal temperature range is 22 degrees C to 37 degrees C. We propose a sequential three-step reactivation process at acidic pH, dimerization of monomers (rate-determining second order step), rapid formation of inactive tetramers, and slow formation of active tetramers (overall rate-determining first order step). Whether reactivation of human beta-tryptase occurs at extracellular or intracellular sites, where the pH is acidic in vivo, should be considered.
在中性pH条件下，人肥大细胞β-色氨酸酶通过肝素稳定在其酶活性四聚体形式，并抵抗生物蛋白酶抑制剂的抑制作用。β-色氨酸酶从肝素解离后，活性四聚体在等渗、中性pH环境中迅速转化为无活性单体。尽管在此转化过程中可能存在可逆过渡态，但一旦形成无活性单体，在中性pH条件下添加肝素无法重新组装活性四聚体。当前研究表明，无活性单体可在酸性pH条件下以不依赖肝素的方式完全重新激活。基于对初始反应速率的分析，由无活性单体形成四聚体和活性酶的各自速率决定步骤分别呈现二级和一级动力学。形成四聚体和重新激活的最佳pH约为6，表明组氨酸残基起关键作用。最佳离子强度当量为160 mM NaCl；最佳温度范围为22℃至37℃。我们提出在酸性pH条件下的一个连续三步重新激活过程，单体二聚化（速率决定二级步骤）、无活性四聚体的快速形成以及活性四聚体的缓慢形成（总体速率决定一级步骤）。应考虑人β-色氨酸酶的重新激活是否发生在体内pH呈酸性的细胞外或细胞内位点。
