Karasaki Y, Ohno M
J Biochem. 1978 Sep;84(3):531-8. doi: 10.1093/oxfordjournals.jbchem.a132157.
The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.
使用Nα-乙酰化芳香族氨基酸甲酯的各种核取代衍生物来检测BPN'和卡尔伯格枯草杆菌蛋白酶[EC 3.4.21.14]的动力学特异性，以便与α-胰凝乳蛋白酶相比绘制它们的疏水结合位点。由于kcat值较大，卡尔伯格酶通常比BPN'酶反应性更强。两种枯草杆菌蛋白酶水解Ac-Tyr(PABz)-OMe（一种在OH官能团带有平面反式对苯偶氮苯甲酰基的酪氨酸衍生物）时Km值最小，这一事实表明这些酶具有比迄今所证明的更扩展的芳香族结合位点。Ac-Phe(4-NO2)-OMe的显著之处在于其水解时具有特别大的kcat值（在pH 7.8时，卡尔伯格枯草杆菌蛋白酶的kcat值为5500±700 s-1）。卡尔伯格枯草杆菌蛋白酶在kcat方面能区分Ac-Phe(4-NO2)-OMe和Ac-Tyr-OMe，但BPN'枯草杆菌蛋白酶不能，这表明前者的特异性位点对取代基大小的微小变化比后者更敏感。Ac-Trp(NCps)-OMe和Ac-Trp(NCps)-OH以竞争性方式与酶的活性位点结合。两种酶之间结合标准自由能的差异可能表明卡尔伯格枯草杆菌蛋白酶的疏水裂缝比BPN'枯草杆菌蛋白酶的疏水裂缝更深和/或更窄。
