丹酰-L-苯丙氨酸和L-苯丙氨酸对小牛肠碱性磷酸酶抑制作用动力学的比较性研究

Kinetic Study on Inhibition Effects of Dansyl-L-Phenylalanine and L-Phenylalanine on Calf Intestinal Alkaline phosphatase

利用紫外分光光度法揭示了丹酰-L-苯丙氨酸对小牛肠碱性磷酸酶的选择性抑制作用,并比较性地研究了丹酰-L-苯丙氨酸和L-苯丙氨酸对小牛肠碱性磷酸酶的动力学抑制过程。结果表明在37℃和碱性磷酸酶工作的最佳pH值(pH10.4)的条件下,丹酰-L-苯丙氨酸与L-苯丙氨酸类似,能够有效抑制小牛肠碱性磷酸酶的活性;利用双倒数曲线拟合,判定丹酰-L-苯丙氨酸与L-苯丙氨酸的抑制类型均为反竞争性抑制,两者的抑制常数Ki均为mmol级。对丹酰-L-苯丙氨酸抑制小牛肠碱性磷酸酶的作用研究,不仅有助于进一步阐明L-苯丙氨酸对组织特异性碱性磷酸酶的选择性抑制机理,而且丹酰基团的存在也为荧光标定碱性磷酸酶的特效抑制剂提供了可能。

To evaluate the inhibition effect of dansyl-L-phenylalanine on calf intestinal alkaline phosphatase （CIAP）, UV-Vis spectrophotometric method was employed. It was found that dansyl-L-phenylalanine can selectively inhibit CLAP. The kinetic inhibition processes of dansyl-L-phenylalanine and L-phenylalanine were comparatively studied. The authors ＇ finding elucidates that at the optimized alkaline pH of alkaline phosphatase （pH 10. 4） and 37 ℃, dansyl-L-phenylalanine can inhibit alkaline phosphatase activity of CIAP efficiently and specifically, similar as L-phenylalanine. Both inhibition types were uncompetitive inhibition resulting from the double reciprocal curve fitting of v versus substrate concentrations, and the inhibition constants Ki of both inhibitors were determined to be 2.3 and 1.1mmol· L^-1 respectively, both of which were at millimolar level. The investigation of the inhibition effect of dansyl modified L-phenylalanine on calf intestinal alkaline phosphatase not only helped get insight into the detailed inhibition mechanism of L-phenylalanine on tissue specific alkaline phosphatase, such as in the case of intestinal alkaline phosphatase, but also provided the possibility to employ fluorescence spectroscopy by labeling the specific inhibitors of alkaline phosphatase with chromophoric groups.