Antonini E, Ascenzi P, Menegatti E, Bortolotti F, Guarneri M
Biochemistry. 1982 May 11;21(10):2477-82. doi: 10.1021/bi00539a029.
Kinetic studies have been carried out with a well-characterized preparation of human urinary (h.u.) kallikrein using chromogenic substrates. Steady-state and pre-steady-state data for h.u. kallikrein catalyzed hydrolysis of N alpha-carbobenzoxy-L-lysine p-nitrophenyl ester (ZLysONp) and of N alpha-carbobenzoxy-L-alanine p-nitrophenyl ester (ZAlaONp) in the presence and absence of ethylamine and acetamidine have been obtained under various conditions and have been analyzed in the framework of the minimum three-step mechanism: (formula see text) The pH dependencies of the kinetic parameters for the hydrolysis of ZLysONp and ZAlaONp in the presence of saturating levels of ethylamine and acetamidine show that at acid pH values (less than or equal to 4) the k3 step (deacylation) is rate limiting in catalysis, whereas for pH values greater than or equal to 6, k2 (acylation) becomes rate limiting. On the other hand, the acylation step is rate limiting in the enzymatic hydrolysis of ZAlaONp over the whole pH range explored. Saturating concentrations of acetamidine increase, more than those of ethylamine, kcat for the hydrolysis of ZAlaONp. The affinity of h.u. kallikrein for acetamidine and ethylamine changes about 5-fold with pH between pH 5 and 3. The pH dependence of the spectral properties of free hu. kallikrein reflects the ionization of a group with a pKa value of 4.45 +/- 0.1. The results point out that, similarly to bovine beta-trypsin, h.u. kallikrein catalysis involves an ionizable group which has a pKa of about 4.5 in the free enzyme and a pKa of about 3.7 in the enzyme bound to cationic substrates or ligands.
利用发色底物，对具有充分表征的人尿激肽释放酶制剂进行了动力学研究。在有和没有乙胺及脒的情况下，在各种条件下获得了人尿激肽释放酶催化水解Nα - 苄氧羰基 - L - 赖氨酸对硝基苯酯（ZLysONp）和Nα - 苄氧羰基 - L - 丙氨酸对硝基苯酯（ZAlaONp）的稳态和预稳态数据，并在最少三步机制的框架内进行了分析：（公式见原文）在乙胺和脒饱和水平存在下，ZLysONp和ZAlaONp水解动力学参数的pH依赖性表明，在酸性pH值（小于或等于4）时，k3步骤（脱酰基）是催化中的限速步骤，而对于pH值大于或等于6，k2（酰化）成为限速步骤。另一方面，在整个探索的pH范围内，ZAlaONp的酶促水解中酰化步骤是限速步骤。脒的饱和浓度比乙胺更能增加ZAlaONp水解的kcat。人尿激肽释放酶对脒和乙胺的亲和力在pH 5和3之间随pH变化约5倍。游离人尿激肽释放酶光谱性质的pH依赖性反映了一个pKa值为4.45±0.1的基团的电离。结果指出，与人β - 胰蛋白酶类似，人尿激肽释放酶催化涉及一个可电离基团，该基团在游离酶中的pKa约为4.5，在与阳离子底物或配体结合的酶中的pKa约为3.7。
