QUANTITATION OF FACTORS WHICH AFFECT HYDROLASE AND TRANSGALACTOSYLASE ACTIVITIES OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI) ON LACTOSE
QUANTITATION OF FACTORS WHICH AFFECT HYDROLASE AND TRANSGALACTOSYLASE ACTIVITIES OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI) ON LACTOSE
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DOI：
10.1021/bi00654a029
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
WALLENFELS, K
中科院分区：
文献类型：
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作者：
HUBER, RE;KURZ, G;WALLENFELS, K
摘要：对以乳糖为底物的/3-半乳糖苷酶(E.Coli)的水解酶和转半乳糖苷酶活性进行了定量研究，并探讨了影响这些酶活性的各种因素。在低乳糖浓度下，半乳糖产量与葡萄糖产量相当。然而，当乳糖浓度高于0.05M时，半乳糖相对葡萄糖的产生率急剧下降，开始产生三糖和四糖(这两种低聚糖的半乳糖/葡萄糖比分别约为2：1和3：1)。至少形成了五种不同的三糖，它们的形成模式表明它们可能同时利用乳糖和别乳糖作为半乳糖受体。在所有乳糖浓度下(别乳糖/葡萄糖的比值约为0.88)，都能产生与葡萄糖成正比的别乳糖。对各种数据的分析，包括非常早期分析的一个反应，表明生产别半乳糖的主要手段(最初也是唯一的方法)是直接用酶将半乳糖从乳糖的葡萄糖部分的4位转移到6位，而不需要事先从酶中释放葡萄糖。它被展示了，^^。虽然乳糖[半乳糖-/3-D-(L-β4)-吡喃葡萄糖苷]是/3-半乳糖苷酶(/3-半乳糖苷半乳糖苷水解酶，EC3.2)的天然底物。1.23)在大肠杆菌中，用这种酶进行的许多研究中只有相对较少的一部分使用乳糖作为底物(Cohn和Monod，1951；Kuby和Lardy，1953；Reithel和Kim，1960；Wallenfels等人，1960a，b；Becker和Evans，1969；Burstein等人，1965；Jobe和Bourcia，1972)。大多数关于/3-半乳糖苷酶的研究都是关于该酶对合成底物邻硝基苯基/3-D-吡喃葡萄糖苷(ONPG1)的水解性作用(Wallenfels and Weil，1972)，而几乎完全忽略了该酶所具有的转半乳糖苷酶的作用。关于/3-半乳糖苷酶相对于水解酶的转半乳糖基酶反应的定量研究以前还没有进行过。因此，以乳糖为底物，对/3-半乳糖苷酶的作用进行全面的定量研究，包括转半乳糖酶活性对总转化率的影响，似乎是有意义的。这似乎特别重要，因为它
R. E. Huber,* G. Kurz, and K. Wallenfels abstract: A study was implemented to quantitate the hydrolase and transgalactosylase activities of/3-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concen-trations therate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concen-trations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2: 1 and 3: 1, respectively, were found for these two types of oligosaccharides). At least five different trisaccharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown,.^^. lthough lactose [galactosyl-/3-D-(l-» 4)-glucopyranose] is known to be the natural substrate of/3-galactosidase (/3-dgalactoside galactohydrolase, EC 3.2. 1.23) of Escherichia coli, only a relatively small number of the many studies carried out with this enzyme haveused lactose as the substrate (Cohn and Monod, 1951; Kuby and Lardy, 1953; Reithel and Kim, 1960; Wallenfels et al., 1960a, b; Becker and Evans, 1969; Burstein et al., 1965; Jobe and Bourgeois, 1972). Mostof the studies on/3-galactosidase have dealt with the hydrolytic action of the enzyme on the synthetic substrate o-nitrophenyl-/3-D-galac-topyranoside (ONPG1)(Wallenfels and Weil, 1972) and have almost totally ignored the transgalactosylase action which the enzyme possesses.[The exception to this are studies in which simple alcohols were tested as acceptors of galactosylmoieties from synthetic substratessuch as ONPG (Shifrin and Hunn, 1969; Wallenfels and Weil, 1972).] Studies quantitating the transgalactosylase reactions of/3-galactosidase relative to the hydrolytic reactions have not previously been done. It, therefore, seemed pertinent to carry out a comprehensive quantitative study of the action of/3-galactosidase using lactose as the substrate, including the effect of transgalactosylase activity on the overall rate. This seemed especially important since it