摘要
三种有机溶剂,二氧六环、二甲基甲酰胺和乙二醇,都不同程度地抑制中华猕猴桃蛋白酶催化水解赖氨酸对硝基本酯(CBZ-LYS-pNP)能力,其动力学行为符合Michaelis-Menten方程式,二氧六环的抑制作用类似于竞争性抑制类型,二甲基甲酰胺和乙二醇为非竞争性抑制,其抑制常数,分别为k_1=5.6×10^(-2)、0.58和1.60mol/L,光谱分析表明,酶在有机溶剂中,空间构象也发生了变化,随着二氧六环和二甲基甲酰胺浓度的增大,酶分子的α-螺旋度下降,无规卷曲度上升,荧光发射强度比天然酶略有增高或降低,变化的规律性不够,紫外差光谱显示,酶在6％二甲基甲酰胺中有一个243nm的负峰,此负峰随浓度的增大而增大,并略向红移,在24％二甲基甲酰胺中,负峰红移至248nm处。
Actinidin, a thiol protease, was isolated and purified from the fruit of actnidia chi nensts. The kinetic studies showed that the hydrolysis reaction catalyzed by actimdin in dioxane, dimethylformamide and ethylene glycol obey Michaelis-Menten kinetics. All three different organic solvents can inhibit the activity of the enzyme. Their inhibition mechanism is similiac to the noncom-petitive type in dioxane but displayed a competitive-like inhibition in dimethylformamide and ethylene glycol. Their respective inhibition constants, k1, are 5. 6×102; 0. 58 and 1. 6 mol/L, at 25℃ pH 4. 0. Changes of the enzyme conformation in the presence of organic solvents were measured by CD, fluorescence and differential absorption spectra. The a helix contents of enzyme decreased with increasing organic solvents concentration, which were consistent with the loss of the enzyme activity; At the same time the fluorescence intensities of the peaks at 329 nm decreased or increased siightly- The difference spectra showed one negative peak at 233 nm in 6% dimethylformamtde. The height of the negative peak increased greatly accompanying a red-shift of the peak position to 248 nm in 24% dimethylformamide.
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
1993年第2期210-215,共6页
Journal of Xiamen University：Natural Science
基金
国家自然科学基金
关键词
猕猴桃
蛋白酶
半极性介质
Actinidin, Organic media, Kinetic study, CD, Differential absorption, Fluorescence spectra
