大麦α-淀粉酶/枯草杆菌蛋白酶抑制剂与高pI大麦α-淀粉酶反应的停流动力学研究。

Stopped-flow kinetic studies of the reaction of barley alpha-amylase/subtilisin inhibitor and the high pI barley alpha-amylase.

作者信息

Sidenius U, Olsen K, Svensson B, Christensen U

机构信息

Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.

出版信息

FEBS Lett. 1995 Mar 20;361(2-3):250-4. doi: 10.1016/0014-5793(95)00187-e.

DOI:10.1016/0014-5793(95)00187-e

PMID:7698332

Abstract

The interaction of alpha-amylase/subtilisin inhibitor (BASI) from barley seeds and the high pI barley alpha-amylase (AMY2) de novo synthesized during seed germination, has been studied at pH 8.0, 25 degrees C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: [formula: see text] The resulting values of the kinetic parameters were: k2/K1 = (1.0 +/- 0.2) x 10(6) M-1.s-1, K1 = 0.4 +/- 0.21 mM, k2 = 320 +/- 150 s-1, k-2 = (7.2 +/- 0.6) x 10(-5)s-1, and the overall dissociation constant Kd = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2.

摘要

利用停流荧光光谱法、平衡荧光滴定法以及对底物蓝色淀粉从BASI-AMY2复合物中置换BASI的动力学分析，在pH 8.0、25℃条件下，对大麦种子中的α-淀粉酶/枯草杆菌蛋白酶抑制剂（BASI）与种子萌发过程中从头合成的高pI大麦α-淀粉酶（AMY2）之间的相互作用进行了研究。结果符合两步反应模型：[公式：见原文]。所得动力学参数值为：k2/K1 = (1.0 ± 0.2) × 10(6) M-1·s-1，K1 = 0.4 ± 0.21 mM，k2 = 320 ± 150 s-1，k-2 = (7.2 ± 0.6) × 10(-5)s-1，总解离常数Kd = (0.7 ± 0.1) × 10(-10) M。因此，BASI的最佳特征是作为AMY2的快速反应、紧密结合抑制剂。