Rahmo A, Fife TH
Department of Biochemistry, University of Southern California, Los Angeles, California
Bioorg Chem. 2000 Aug;28(4):226-241. doi: 10.1006/bioo.2000.1161.
N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxy-peptidase A; at 30 degrees C and pH 7.5, K(m) is 2.6 x 10(-5) M while k(cat) is 177 s(-1) (k(cat)/K(m) = 6.8 x 10(6) M(-1) s(-1)). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of N-benzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (beta = 0.18). Employing the ester substrate hippuryl-DL,beta-phenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-L-phenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (beta = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S'(1) site, while N-benzoyl-L-phenylalanine binds in the S(1)-S(2) site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding. Copyright 2000 Academic Press.
N-苯甲酰基-L-苯丙氨酰-L-苯丙氨酸是羧肽酶A的一种优良肽底物；在30℃和pH 7.5条件下，米氏常数（K(m)）为2.6×10⁻⁵ M，催化常数（k(cat)）为177 s⁻¹（k(cat)/K(m) = 6.8×10⁶ M⁻¹ s⁻¹）。吲哚-3-乙酸是该肽和马尿酸-L-苯丙氨酸的非竞争性或混合型抑制剂；E/V对[抑制剂]的作图呈线性。N-苯甲酰基-L-苯丙氨酸是肽水解的竞争性抑制剂，E/V对[抑制剂]的作图同样呈线性。每个活性位点结合一分子抑制剂，且这些抑制剂结合在不同位点。在恒定肽底物浓度和一系列恒定吲哚-3-乙酸浓度下，E/V对N-苯甲酰基-L-苯丙氨酸浓度的作图呈线性，且在E/V轴后方和[抑制剂]轴上方相交。这表明两种抑制剂可同时结合，且一种抑制剂的结合促进另一种抑制剂的结合（β = 0.18）。使用酯底物马尿酸-DL，β-苯乳酸时，观察到相反类型的行为；N-苯甲酰基-L-苯丙氨酸是线性非竞争性抑制剂，吲哚-3-乙酸是线性竞争性抑制剂。同样，两种抑制剂的作图呈线性，且在[抑制剂]轴上方相交（β = 0.12）。先前的X射线晶体学研究表明，吲哚-3-乙酸结合在S'(1)位点的疏水口袋中，而N-苯甲酰基-L-苯丙氨酸结合在S(1)-S(2)位点。N-苯甲酰基-L-苯丙氨酰-L-苯丙氨酸水解的产物复合物（苯丙氨酸 + N-苯甲酰基-L-苯丙氨酸）占据这两个位点。然而，目前的研究表明，在pH 7.5时，肽底物与酶的结合方式使其不会与吲哚-3-乙酸竞争。结合位点可能是通过底物和产物结合诱导或稳定的构象变化形成的。版权所有2000年学术出版社。
