Stein R L, Strimpler A M
Biochemistry. 1987 Apr 21;26(8):2238-42. doi: 10.1021/bi00382a025.
Acyl-enzymes of human leukocyte elastase (HLE) were generated in situ during the hydrolysis of peptide thiobenzyl esters and served as substrates for aminolysis by a variety of amino acid amides and short peptide nucleophiles. For amino acid amides, there is a positive correlation between nucleophilic reactivity toward N-methoxysuccinyl (MeOSuc)-Ala-Ala-Pro-Val-HLE and the hydrophobicity of the side chain. For peptides, nucleophilicity toward MeOSuc-Ala-Ala-Pro-Val-HLE decreases dramatically with increasing chain length. Combined, these results suggest that substrate specificity for the P1' residue may be more dependent on side chain hydrophobicity than on specific, structural features of the side chain and there may be no important binding interactions available past S1'. Kinetic parameters were also determined for the nucleophilic reactions of PheNH2 and TyrNH2 with MeOSuc-Pro-Val-HLE, MeOSuc-Ala-Pro-Val-HLE, MeOSuc-Ala-Ala-Pro-Val-HLE, and MeOSuc-Ala-Ala-Pro-Ala-HLE. Reactivity of these acyl-enzymes toward nucleophilic attack displays no dependence on peptide chain length but does increase significantly for the substrate with Ala at P1. This same correlation between reactivity and acyl-enzyme structure is also seen for nucleophilic attack by water.
在肽硫代苄酯水解过程中原位生成了人白细胞弹性蛋白酶（HLE）的酰基酶，这些酰基酶可作为多种氨基酸酰胺和短肽亲核试剂进行氨解反应的底物。对于氨基酸酰胺，其对N - 甲氧基琥珀酰（MeOSuc）-Ala - Ala - Pro - Val - HLE的亲核反应活性与侧链疏水性之间存在正相关。对于肽类，随着链长增加，其对MeOSuc - Ala - Ala - Pro - Val - HLE的亲核性急剧下降。综合这些结果表明，P1'残基的底物特异性可能更多地取决于侧链疏水性，而非侧链的特定结构特征，并且在S1'之后可能不存在重要的结合相互作用。还测定了PheNH2和TyrNH2与MeOSuc - Pro - Val - HLE、MeOSuc - Ala - Pro - Val - HLE、MeOSuc - Ala - Ala - Pro - Val - HLE以及MeOSuc - Ala - Ala - Pro - Ala - HLE亲核反应的动力学参数。这些酰基酶对亲核攻击的反应性不依赖于肽链长度，但对于P1位为Ala的底物，反应性显著增加。对于水的亲核攻击，反应性与酰基酶结构之间也存在相同的相关性。
