Tellam R, de Jersey J, Winzor D J
Biochemistry. 1979 Nov 27;18(24):5316-21. doi: 10.1021/bi00591a009.
The binding of N-acetyl-tryptophan to the monomeric and dimeric forms of alpha-chymotrypsin in I = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 L/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric alpha-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/L) alpha-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of alpha-chymotrypsin.
在离子强度I = 0.2的醋酸盐 - 氯化物缓冲液（pH 3.86）中，对N - 乙酰 - 色氨酸与α - 胰凝乳蛋白酶单体和二聚体形式的结合进行了定量研究。在没有抑制剂的情况下进行的平衡沉降研究得出二聚化常数为3.5 L/g。该值通过在Bio - Gel P - 30上对该酶进行前沿凝胶色谱分析得到证实，该方法还用于确定N - 乙酰 - L - 色氨酸优先与单体酶结合。通过以N - 乙酰 - L - 色氨酸乙酯为底物的竞争性抑制动力学研究，确定N - 乙酰 - L - 色氨酸与单体α - 胰凝乳蛋白酶结合的平衡常数为1300 M⁻¹。根据这些结果以及用浓缩（18.5 g/L）α - 胰凝乳蛋白酶获得的结合数据，计算出与二聚体酶相应相互作用的固有结合常数为250 M⁻¹。目前的结果驳斥了早期关于竞争性抑制剂仅与单体结合的说法，以及关于抑制剂与α - 胰凝乳蛋白酶单体和二聚体形式结合等效性的说法。
