Huber R E, Roth N J, Bahl H
Division of Biochemistry, Faculty of Science, University of Calgary, Canada.
J Protein Chem. 1996 Oct;15(7):621-9. doi: 10.1007/BF01886744.
The beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the alpha-peptide and an important alpha-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the beta-galactosidase from Escherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity with o-nitrophenyl-beta-D-galactopyanoside (ONPG) was very similar to that with p-nitrophenyl-beta-D-beta-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to the E.coli beta-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by the T. thermosulfurigenes beta-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of the E. coli beta-galactosidase. Trp-999 is probably of the most importance. Trp-999 of the E. coli beta-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of the T. thermosulfurigenes beta-galactosidase is different was strengthened by competitive inhibition studies. Compared to E. coli beta-galactosidase, D-galactonolactone was a very good inhibitor of the T. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate that T. thermosulfurigenes beta-galactosidase binds the transition state differently than does E. coli beta-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature of T. thermosulfurigenes.
已发现来自嗜热栖热硫化叶菌EM1的β-半乳糖苷酶是一种二聚体，单体分子量约为85,000。它缺少二聚体-二聚体相互作用所需的α-肽和一个重要的α-螺旋，并且在其他重要的二聚体-二聚体相互作用区域没有同源性。这些结构上的差异可能解释了其二聚体（而非四聚体）结构。每个单体仅结合0.19 Mg2+，并且Mg2+对活性和热稳定性的影响很小。缺少与大肠杆菌β-半乳糖苷酶中Mg2+的三个配体中的两个（Glu-416和His-418）等效的残基，可能解释了Mg2+结合水平较低以及随之而来的对Mg2+缺乏反应的原因。Na+和K+对活性也均无影响。该酶对邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）的活性与对对硝基苯基-β-D-吡喃半乳糖苷（PNPG）的活性非常相似，并且ONPG的pH谱与PNPG的pH谱非常相似。这些差异与大肠杆菌β-半乳糖苷酶形成对比，后者对这两种底物有明显区分。嗜热栖热硫化叶菌β-半乳糖苷酶缺乏区分能力可能是由于缺少与大肠杆菌β-半乳糖苷酶910-1023位残基等效的序列。Trp-999可能最为重要。大肠杆菌β-半乳糖苷酶的Trp-999对于糖苷配基结合很重要，而ONPG和PNPG仅在其糖苷配基上有所不同。竞争性抑制研究进一步证明了嗜热栖热硫化叶菌β-半乳糖苷酶的糖苷配基位点不同的观点。与大肠杆菌β-半乳糖苷酶相比，D-半乳糖内酯是嗜热栖热硫化叶菌酶的一种非常好的抑制剂，而L-核糖的抑制作用较弱。这些都是过渡态类似物，结果表明嗜热栖热硫化叶菌β-半乳糖苷酶与大肠杆菌β-半乳糖苷酶结合过渡态的方式不同。甲醇和葡萄糖是半乳糖的良好受体，当葡萄糖作为受体时会形成别乳糖。然而，当乳糖作为底物时，用薄层层析法无法检测到别乳糖。所指出的这些差异可能归因于嗜热栖热硫化叶菌的嗜热性质。
