A TEMPERATURE JUMP STUDY OF ASPARTATE AMINOTRANSFERASE . A REINVESTIGATION
A TEMPERATURE JUMP STUDY OF ASPARTATE AMINOTRANSFERASE . A REINVESTIGATION
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DOI：
10.1021/bi00858a031
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发表时间：
1967-01-01
期刊：
影响因子：
2.9
通讯作者：
HAMMES, GG
中科院分区：
文献类型：
--
作者：
FASELLA, P;HAMMES, GG
用温度跳跃技术研究了天冬氨酸氨基转移酶α亚型与底物的相互作用。三个松弛时间与酶和每个氨基酮酸对、谷氨酸-酮戊二酸和天冬氨酸-草乙酸酯的反应有关。这意味着在每个半‘-反应中至少存在两个反应中间体；由于光谱证据表明存在甚至超过两个中间体，动力学结果从涉及任意数量的中间体的机理的角度进行了讨论。每个半反应的最长驰豫时间与浓度的依赖关系允许确定特定的速率和结合常数。不能从其他驰豫时间获得单独的速率常数，但与涉及氨基酸的双分子和解离过程有关的速率常数的下限可以估计为10-108M-1秒-1和105-106秒-1，相应的酮酸下限为103M-1秒-1和104秒-1。还研究了300-500微米范围内弛豫效应幅度与波长的关系。结果表明，在每个HA IF反应中，具有360、430和490m[u]光谱峰的中间体出现在最慢的步骤的一侧，而在330 m[u]处有一个光谱峰。与另一边的中间体有关。用温度跳跃法得到的数据与用分光光度法测得的平衡常数和稳态动力学研究的结果是一致的。
The interaction of the a subform of aspartate aminotransferase with its substrates was studied with the temperature jump technique. Three relaxation times are associated with the reaction of the enzyme and each amino-keto acid pair, glutamate-ketoglutarate and aspartate-oxalacetate. This implies at least 2 reaction intermediates exist in each half''-reaction; since spectral evidence suggests even more than 2 intermediates are present, the kinetic results are discussed in terms of a mechanism involving an arbitrary number of intermediates. The concentration dependence of the longest relaxation time for each half-reaction permits determination of specific rate and binding constants. Individual rate constants could not be obtained from the other relaxation times, but lower bounds of 10 -108 M-1 sec-1 and 105-106 sec-1 can be estimated for the rate constants associated with the bimolecular and dissociation processes involving amino acids; the corresponding lower bounds for the keto acids are 103 M-1 sec-1 and 104 sec-1. The wavelength dependence of the amplitudes of the relaxation effects over the range 300-500 mu was also investigated. The results obtained suggest intermediates with spectral peaks at 360, 430, and 490 m[mu] occur on one side of the slowest step in each ha If-reaction, while a spectral peak at 330 m[mu]. is associated with intermediates on the other side. The data obtained with the temperature jump method are consistent with equilibrium constants measured spectrophotometrically and with the results of steadystate kinetic studies.