摘要
发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10^(-4)mol/L,k_(cat)为742min^(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。
A synthetic small substrate, CBZ-Lys·pNP, was found to be hydrolyzed by fruit bromelain. The Michaelis constant(Km) was 4.167 × 10-4(mol/L) ,and the kcat 742 (rain-1), at 25℃ pH5.6. Changes of the enzyme conformation in the presence of guanidine and SDS were measured by fluorescence and differential UV-absorption spectra. The fluorescence intensity of the enzyme gradually decreased with increasing guanidine concentrations. The fluorescence emission peak (at 332 nm) of the enzyme in 4mol/L guanidine solution was red-shifted to 353nm, and a new fluorescence emission peak appeared at 310nm. The fluorescence emission peak intensity of the enzyme decreased under SDS denaturation. When the SDS concentration was greater than 3.47mmol/L,the fluorescence emission peak of the enzyme increased again in intensity and was red-shifted in position, and a new fluorescence emission shoulder appeared at 315nm. Differential UV-absorption spectra of the enzyme SDS solution at appeared different concentrations showed that there was a rather distinct negative peak at 236nm, which is in accord with the change of the α-helix structure of the enzyme. There were also three negative peaks at 278nm, 286nm and 295nm, which indicate that the microenviron-ments of tyrosine,tryptophan and phenylalanine in the enzyme molecule had obviously changed. Denaturation and inactivation rate constants of the enzyme were measured respectively in different concentrations of SDS and guanidine solution. Changes in enzyme conformation and catalytic activity were compared. In the presence of guanidine and SDS, the rate constants of inactivation were greater than those of denaturation.
基金
国家自然科学基金
关键词
菠萝
蛋白酶
构象
活力
Fruit bromelain
Denaturation and inactivation
Conformation and activity
