Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli)
Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli)
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DOI：
10.1139/o10-009
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发表时间：
2010-10-01
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影响因子：
--
通讯作者：
Huber, Reuben E.
中科院分区：
文献类型：
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作者：
Dugdale, Megan L.;Dymianiw, Dayna L.;Huber, Reuben E.
β -半乳糖苷酶的Met - 542残基对酶的活性很重要，因为它作为Phe - 601的苄基侧链在两个稳定位置之间移动的导向。这种移动与一个活性位点环（残基794 - 803）的重要构象变化（开放与闭合）协同发生。Phe - 601和Arg - 599通过Phe - 601的π电子和Arg - 599的胍阳离子相互作用，当Met - 542被Ala残基取代时，由于导向作用缺失，它们移出正常位置并变得无序。由于Phe - 601的主链羰基是Na⁺的一个配体，Na⁺也移出正常位置并变得无序；Na⁺的结合能力降低约120倍。反过来，另外两个Na⁺配体Asn - 604和Asp - 201也变得无序。一种底物类似物（IPTG）使Arg - 599、Phe - 601和Na⁺恢复到它们正常的开环位置，而一种过渡态类似物（D - 半乳糖内酯）使它们恢复到正常的闭环位置。这些化合物也使Phe - 601、Asn - 604、Asp - 201和Na⁺恢复有序。然而，需要结合能来恢复结构和秩序。oNPG和pNPG的Ks值以及底物类似物的竞争Ki值比天然酶高90 - 250倍，而过渡态类似物的竞争Ki值高约3.5 - 10倍。因此，E×S能级比E×过渡态能级升高得更多，半乳糖基化所需的活化能更少。所以M542A - β - 半乳糖苷酶的半乳糖基化速率（k₂）增加。然而，去半乳糖基化速率（k₃）降低，因为E×过渡态复合物稳定性降低。
The Met-542 residue of beta-galactosidase is important for the enzyme's activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794-803). Phe-601 and Arg-599, which interact with each other via the p electrons of Phe-601 and the guanidium cation of Arg-599, move out of their normal positions and become disordered when Met-542 is replaced by an Ala residue because of the loss of the guide. Since the backbone carbonyl of Phe-601 is a ligand for Na(+), the Na(+) also moves out of its normal position and becomes disordered; the Na(+) binds about 120 times more poorly. In turn, two other Na(+) ligands, Asn-604 and Asp-201, become disordered. A substrate analog (IPTG) restored Arg-599, Phe-601, and Na(+) to their normal open-loop positions, whereas a transition state analog (D-galactonolactone) restored them to their normal closed-loop positions. These compounds also restored order to Phe-601, Asn-604, Asp-201, and Na(+). Binding energy was, however, necessary to restore structure and order. The K(s) values of oNPG and pNPG and the competitive K(i) values of substrate analogs were 90-250 times higher than with native enzyme, whereas the competitive K(i) values of transition state analogs were similar to 3.5-10 times higher. Because of this, the E circle times S energy level is raised more than the E circle times transition state energy level and less activation energy is needed for galactosylation. The galactosylation rates (k(2)) of M542A-beta-galactosidase therefore increase. However, the rate of degalactosylation (k(3)) decreased because the E circle times transition state complex is less stable.