摘要
巳纯化的溜曲霉β-N-乙酰氨基己糖苷酶(表现出β-N-乙酰氨基葡萄糖苷酶和β-N-乙酰氨基半乳糖苷酶活力)水解对硝基酚-N-乙酰氨基葡萄糖苷和对硝基酚-N-乙酰氨基半乳糖苷,其 K_m 值分别为0.44和2.0mmol/L。V_(max)值分别为740.0和476.5μmol·min^(-1)·mg^(-1)。氨基葡萄糖、氨基半乳糖、GlcNAc 及 GalNAc 均为这两个酶的竞争性抑制剂,对β-GlcNAcase的 K_1 值分别为34.9、62.5、16.9及38.2mmol/L,而对β-GalNAcase 的 K_1值分别为24.1、75.0、50.0及131.8mmol/L。将 pNPGlcNAc 和 pNPGalNAc 等量混合为底物,其酶活力小于单独以 pNPGlcNAc 为底物的活力,大于以 pNPGalNAc 为底物的活力。两种底物以不同摩尔分数混合测活结果,用 Lineweaver-Burk 双倒数法作图,均为直线,求出表观 V′_(max),将各个 V′_(max) 对摩尔分数 f(从 f=0至 f=1)作图,没有出现最大值。由抑制作用及混合底物竞争动力学说明β-GlcNAcase 和β-GalNAcase 同存在于一个蛋白质,并共用一个活力部位。
The purified β-N-acetylhexosaminidase from Asp.tamar hydrolyzes bothp-nitrophenyl β-N-Acetvlglucosaminide and p-nitrophenyl β-N-Acetylgalacto(?)a-minide.The enzyme has K_m of 0.44 and 2.0mmol/L and V_(max) values of 740.0 and476.5μmol·min^(-1)·mg^(-1) for the two substrates respectively.Glucosamine,gaia-ctosamine,GlcNAc and GalNAc competitively inhibited both enzymatic activities(?)the K_i values of the inhibitors to β-GlcNAcase were 34.9,62.5,19.9 and 38.2mmol/Lrespectively.,The K_i values of the inhibitors to β-GalNAcase were 24.1,750.50.0 and 131.8mmol/L respectively.The enzyme activities toward pNPGIcNAc,pNPGalNAc,and mixtures were determined and activities toward mixtures werelower than that toward pNPGlcNAc and higher than that toward pNPGalNAc.Whentesting the two substrates mixtures in different molar fractions,the results obtainedby Lineweaver-Burk plote showed linear relation.The diagram of apparent V'_(?)versus the mole fractions showed a gradual change between f=1 and f=0 withoutmaximum.From competitive inhibition of enzyme by products and analogues andfrom the competition between the two substrates,it may be concluced that β-GlcNAcase and β-GalNAcase activities were catalyzed by a common active site.
出处
《微生物学报》
CAS
CSCD
北大核心
1992年第6期405-411,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金
中国科学院资助项目
关键词
溜曲霉
糖苷酶
Aspergillus tamarii
β-N-Acetylhexosaminidase
Glycosidase
A common active site for two activities
