Krishnaswamy S, Bryan J K
Arch Biochem Biophys. 1983 Nov;227(1):210-24. doi: 10.1016/0003-9861(83)90364-8.
The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.
从玉米（Zea mays L.）幼苗中分离出的苏氨酸敏感型高丝氨酸脱氢酶（L-高丝氨酸：NAD(P)+氧化还原酶）具有可变的动力学和调节特性。此前对该酶的分析表明，它能够在四种动力学上不同的状态之间进行配体介导的相互转化（S. Krishnaswamy和J. K. Bryan（1983年），《生物化学与生物物理学档案》222卷，449 - 463页）。已鉴定出这些酶的形式，并发现它们在寡聚体构型和构象上存在差异。在KCl和苏氨酸存在的情况下，酶的三种形式（B、T和K）之间建立了快速平衡。在测定条件下，这些形式中的每一种都能经历独特的缓慢转变为稳态形式的过程。凝胶过滤色谱法和蔗糖密度离心法得到的结果表明，B形式和稳态形式是四聚体，T和K状态是二聚体。有证据表明，从一种二聚体形式到另一种二聚体形式的快速转化只能通过形成四聚体B状态来发生。在反应酶条件下进行色谱分析为从酶的任何其他形式缓慢形成共同的稳态形式提供了直接支持。转变速率受苏氨酸、高丝氨酸、NAD+的影响，对于涉及缔合反应的转变，还受酶浓度的影响。T和K形式以及B和稳态形式在表观大小上存在微小的、可重复的差异，这归因于构象的变化。这一结论得到了酶状态对蛋白水解失活的不同敏感性、二硫代双硝基苯甲酸不同失活速率以及它们热稳定性改变的支持。此外，酶的B、T和K状态表现出独特的内在荧光光谱。光谱变化与酶的动力学和滞后特性的变化密切平行。多种分析方法的结果在内部是一致的，并为这一结论提供了相当多的支持，即这种多效调节酶可以以几种物理上不同的状态中的任何一种存在。
