Roth N J, Huber R E
Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Biol Chem. 1996 Jun 14;271(24):14296-301. doi: 10.1074/jbc.271.24.14296.
beta-Galactosidases with substitutions for His-540 were only poorly reactive with galactosyl substrates. However, the activity with substrates that were like galactose but did not have a C6 hydroxyl group was not decreased much as a result of such substitutions. The loss of transition state stabilization for galactosyl substrates as a result of substitution was between -15.4 and -22.8 kJ/mol but only between +0.34 and -6.5 for substrates that were identical to galactose but lacked the C6 hydroxyl. These findings indicate that an important function of His-540 is to aid in the stabilization of the transition state by forming a stable interaction with the C6 hydroxyl group. This suggestion was strengthened by the results of competitive inhibition studies showing that L-arabinolactone (a transition state analog inhibitor of beta-galactosidase without a C6 hydroxymethyl group) was bound as well by the substituted enzymes as by wild type, whereas transition state analog inhibitors that contain C6 hydroxyls (L-ribose and D-galactonolactone) were bound much more poorly by the substituted enzymes than by the wild type enzyme. Substrate analog inhibitor studies showed that His-540 was also important for binding interactions with the C6 hydroxyl group of the ground (substrate) state. The activation by Mg2+ was the same for the substituted enzymes as for the wild type, and equilibrium dialysis showed that H540F-beta-galactosidase bound Mg2+ as well as did normal beta-galactosidase. The k2 and Ks values seem to have the same pH interactions as wild type enzyme, whereas the k3 interactions are affected differently by pH in the substituted enzymes than in the wild type enzyme. The rate of the "degalactosylation" reaction was affected more by substitutions for His-540 than was the rate of the "galactosylation" reaction. All three substituted beta-galactosidases were less stable to heat than was wild type, but H540N-beta-galactosidase was somewhat more stable than the other two substituted enzymes. There were some differences in activity and inhibitory properties that resulted from the different substitutions.
组氨酸540被取代的β-半乳糖苷酶与半乳糖基底物的反应性很差。然而，对于那些类似于半乳糖但没有C6羟基的底物，这种取代导致的活性下降并不多。取代导致半乳糖基底物的过渡态稳定性损失在-15.4至-22.8 kJ/mol之间，但对于与半乳糖相同但缺少C6羟基的底物，仅在+0.34至-6.5之间。这些发现表明，组氨酸540的一个重要功能是通过与C6羟基形成稳定的相互作用来帮助稳定过渡态。竞争性抑制研究结果强化了这一观点，该研究表明L-阿拉伯糖内酯（一种没有C6羟甲基的β-半乳糖苷酶的过渡态类似物抑制剂）被取代酶和野生型酶结合的程度相同，而含有C6羟基的过渡态类似物抑制剂（L-核糖和D-半乳糖内酯）被取代酶结合的程度比野生型酶差得多。底物类似物抑制剂研究表明，组氨酸540对于与基态（底物）的C6羟基的结合相互作用也很重要。取代酶的Mg2+激活与野生型相同，平衡透析表明H540F-β-半乳糖苷酶结合Mg2+的情况与正常β-半乳糖苷酶相同。k2和Ks值似乎与野生型酶具有相同的pH相互作用，而k3相互作用在取代酶中受pH的影响与野生型酶不同。“去半乳糖基化”反应的速率比“半乳糖基化”反应的速率受组氨酸540取代的影响更大。所有三种取代的β-半乳糖苷酶的热稳定性都比野生型差，但H540N-β-半乳糖苷酶比其他两种取代酶稍微稳定一些。不同的取代导致了活性和抑制特性的一些差异。
