STEADY-STATE KINETIC MECHANISM OF BOVINE BRAIN TUBULIN - TYROSINE LIGASE
STEADY-STATE KINETIC MECHANISM OF BOVINE BRAIN TUBULIN - TYROSINE LIGASE
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DOI：
10.1042/bj2860243
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发表时间：
1992-08-15
影响因子：
4.1
通讯作者：
PURICH, DL
中科院分区：
文献类型：
--
作者：
DEANS, NL;ALLISON, RD;PURICH, DL
研究了酪氨酸和未酪氨酸化的微管蛋白的atp依赖性再合成微管蛋白，建立了微管蛋白最可能的稳态动力学机制：酪氨酸连接酶（adp形成）。三组成对初始速率实验，包括两个底物成对变化，第三个底物保持在高（但不饱和）水平，产生收敛线数据，这种行为是序列机制的诊断。ATP、未酪氨酸化微管蛋白和l -酪氨酸的Michaelis常数分别为14、1.9和17 μ m，最大流速为0.2 pm/min。AMP对ATP是竞争性抑制剂，对微管蛋白或酪氨酸是非竞争性抑制剂。同样，l -二羟基苯丙氨酸相对于酪氨酸具有竞争性，而相对于ATP或微管蛋白具有非竞争性。这些发现直接支持随机顺序机制。ADP的产物抑制模式也与这一分配一致；然而，对正磷酸盐或酪氨酸化微管蛋白的抑制研究并不实际，因为两者都是非常弱的抑制剂。底物保护酶免受n-乙基马来酰亚胺的烷基化和热失活，以及酶与ATP Sepharose和微管蛋白-Sepharose结合的证据，也支持了这种三底物酶反应表现出随机底物添加的观点
The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14-mu-M, 1.9-mu-M and 17-mu-M for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 pm/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition