Pócsi I, Taylor S A, Richardson A C, Smith B V, Price R G
Institute of Biochemistry, L. Kossuth University, Debrecen, Hungary.
Biochim Biophys Acta. 1993 Apr 21;1163(1):54-60. doi: 10.1016/0167-4838(93)90278-y.
The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli beta-D-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed to interactions not involving the catalytic site. When the product of the maximum observed velocity (Vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.
使用五种新开发的比色底物研究了来自牛肝和睾丸、大肠杆菌、黑曲霉和刀豆的β-半乳糖苷酶的动力学特性。酶水解释放的所有发色团在光谱的可见光区域都有高消光系数。使用这些底物（VBzTM-Gal、VLM-Gal、VLPr-Gal、VQM-Gal和VQPr-Gal）中的每一种都发现了不同程度的底物抑制，但如果使用正确的测定条件，这并不是一个重大问题。这些底物与大肠杆菌β-D-半乳糖苷酶的活性中心结合特别紧密，导致低Km值。数据表明，糖苷配基中远离糖苷氧的杂环部分的化学性质不影响底物特异性，底物抑制可归因于不涉及催化位点的相互作用。当计算每种底物的最大观察速度（Vm）与摩尔吸收系数的乘积时，可以评估底物用于每种酶测定的相对优点。来自真菌和细菌来源的β-D-半乳糖苷酶最有效地水解底物，表明它们在分子生物学和生物技术领域可能具有特殊价值。
