Turnbull J, Morrison J F
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Biochemistry. 1990 Nov 6;29(44):10255-61. doi: 10.1021/bi00496a015.
The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.
已经研究了底物类似物、终产物酪氨酸和蛋白质修饰剂碘乙酸对双功能酶分支酸变位酶-预苯酸脱氢酶的抑制作用。这些研究的目的是确定该酶催化的两个反应是在单个活性位点还是在两个独立的活性位点发生。支持变位酶和脱氢酶反应在两个相似但不同的活性位点催化这一结论的证据来自以下结果：（1）竞争性抑制变位酶反应的底物类似物（内氧杂双环二酸）对脱氢酶反应没有影响。（2）丙二酸及其几种衍生物在变位酶反应中作为分支酸的抑制类似物，在脱氢酶反应中作为预苯酸的抑制类似物。然而，通过对变位酶和脱氢酶反应的研究，得到了它们与游离酶相互作用的不同解离常数。（3）在NAD存在下，酪氨酸对变位酶反应的抑制动力学与脱氢酶反应的抑制动力学不同。结果证实，每个亚基用碘乙酸进行羧甲基化会消除变位酶和脱氢酶的活性，并表明酶活性的失活是由于碘乙酸作为活性位点定向抑制剂起作用。
