Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, AL, 35487, United States.
Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, AL, 35487, United States.
Arch Biochem Biophys. 2019 Mar 30;664:1-8. doi: 10.1016/j.abb.2019.01.021. Epub 2019 Jan 19.
The use of modular domains in proteins affords nature a simple route to the diversification of protein function, but co-evolution between domains can complicate large-scale functional annotation. The LeuA dimer regulatory domain is primarily responsible for allosteric feedback inhibition of the enzymes isopropylmalate synthase (IPMS) and citramalate synthase (CMS). In addition to this regulatory role, presence of the domain may also affect substrate selectivity in certain members of the enzyme family. To assess the role of the LeuA dimer regulatory domain in substrate selectivity, truncated versions of IPMS and CMS from Methanococcus jannaschii (MjIPMS and MjCMS, respectively) have been created that lack the LeuA dimer regulatory domain. In the case of MjIPMS, loss of the regulatory domain does not affect substrate selectivity, consistent with previous reports identifying conserved active site residues that play this role. Loss of the regulatory domain in MjCMS, however, results in increased functional promiscuity. Both truncated enzymes exhibit a shift in quaternary structure from tetrameric to monomeric forms as judged by size-exclusion chromatography. Kinetic isotope effects reveal that loss of the regulatory domain results in unique effects on catalysis with chemistry becoming more rate-determining in MjIPMS and less rate-determining in MjCMS. Finally, substitution of conserved active site residues in the promiscuous truncated MjCMS affect substrate selectivity while identical substitutions cause no changes in the wild-type enzyme. Overall, the data predicts a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains.
蛋白质中模块结构域的使用为自然提供了一种简单的途径，可实现蛋白质功能的多样化，但结构域之间的共同进化可能会使大规模的功能注释复杂化。亮氨酸 A 二聚体调节结构域主要负责异丙基苹果酸合酶 (IPMS) 和柠檬酸合酶 (CMS) 的变构反馈抑制。除了这种调节作用外，该结构域的存在也可能影响酶家族中某些成员的底物选择性。为了评估亮氨酸 A 二聚体调节结构域在底物选择性中的作用，分别从詹氏甲烷球菌 (MjIPMS 和 MjCMS) 中创建了缺乏亮氨酸 A 二聚体调节结构域的 IPMS 和 CMS 的截断形式。对于 MjIPMS，调节结构域的缺失并不影响底物选择性，这与先前鉴定出发挥此作用的保守活性位点残基的报道一致。然而，在 MjCMS 中，调节结构域的缺失导致功能变异性增加。两种截断酶都表现出四级结构从四聚体到单体形式的转变，这可以通过凝胶过滤色谱法判断。动力学同位素效应表明，调节结构域的缺失导致催化作用的独特影响，在 MjIPMS 中，化学作用变得更具速率决定性，而在 MjCMS 中则不太具有速率决定性。最后，在变构的截断 MjCMS 中保守活性位点残基的取代会影响底物选择性，而相同的取代在野生型酶中不会引起任何变化。总体而言，数据预测亮氨酸 A 二聚体调节结构域在底物选择性中通过催化调节而不是通过广泛的催化和调节结构域之间的共同进化导致的差异结合来发挥更复杂的作用。
