Petkov D, Christova E, Pojarlieff I, Stambolieva N
Eur J Biochem. 1975 Feb 3;51(1):25-32. doi: 10.1111/j.1432-1033.1975.tb03902.x.
The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase-catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure-enzyme activity studies. Derivatives of alpha-N-acetyl-L-lysine anilide with high sigma minus-value substituents in the aniline ring were synthesized. Rate constants kappa-cat. and apparent Michaelis-Menten constants K-m (app.) are presented. From the substituent dependence of kappa-cat. and from the fact that kappa-cat. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate-limiting step proceeds prior to deacylation. The catalytic rate constant kappa-cat. obeys a linear free-energy relationship of the Hammett type with Q equals +0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid-base catalysed hydrolysis of N-acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low Q-value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate-limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.
由于不存在非生产性结合和底物活化现象，且底物具有良好的溶解性，使得尿激酶催化特定苯胺类化合物的水解反应非常适合用于底物结构-酶活性研究。合成了在苯胺环上带有高σ-负值取代基的α-N-乙酰-L-赖氨酸苯胺衍生物。给出了催化速率常数κ-cat.和表观米氏常数K-m（app.）。根据κ-cat.对取代基的依赖性以及κ-cat.比脱酰化速率常数小13至37倍这一事实，可以得出限速步骤发生在脱酰化之前。催化速率常数κ-cat.服从哈米特类型的线性自由能关系，Q值为+0.72。为了解释这一正的低Q值，提出了由模型反应（在极端条件下N-乙酰甘氨酸苯胺的特定碱催化和一般酸碱催化水解）得到的结果所暗示的两种不同机制。在第一种机制中，酶四面体中间体的分解是限速步骤，而在第二种机制中，其形成控制总速率。然而，无法区分这两种机制。
