Roth N J, Huber R E
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Biochem Biophys Res Commun. 1996 Feb 6;219(1):111-5. doi: 10.1006/bbrc.1996.0190.
Glu-416 of beta-galactosidase (E. coli) was replaced with Gln and Val using site-directed mutagenesis. The substituted enzymes displayed a greatly decreased sensitivity to Mg2+. Equilibrium dialysis studies indicated that wild-type beta-galactosidase bound Mg2+ tightly, whereas E416V-beta-galactosidase did not. In addition, the pH profile of E416V-beta-galactosidase was unaffected by the presence or absence of 1 mM Mg2+. Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+ but high amounts of Mg2+ were needed (1 mM). E416Q-beta-galactosidase was unstable when stored in the presence of Mg2+. The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog inhibitor) in both the presence and the absence of Mg2+.
利用定点诱变技术，将大肠杆菌β-半乳糖苷酶的第416位谷氨酸替换为谷氨酰胺和缬氨酸。取代后的酶对Mg2+的敏感性大幅降低。平衡透析研究表明，野生型β-半乳糖苷酶紧密结合Mg2+，而E416V-β-半乳糖苷酶则不然。此外，E416V-β-半乳糖苷酶的pH谱不受1 mM Mg2+存在与否的影响。令人惊讶的是，两种取代酶都被Mg2+灭活而非激活，但需要大量的Mg2+（1 mM）。当在Mg2+存在下储存时，E416Q-β-半乳糖苷酶不稳定。在有和没有Mg2+的情况下，取代后的酶对合成底物ONPG和IPTG（一种底物类似物抑制剂）的亲和力都显著降低。
