Biphasic inactivation of procine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.
Biphasic inactivation of procine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.
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5-磷酸吡哆醛对猪心脏线粒体苹果酸脱氢酶的双相失活。
DOI：
10.1016/s0021-9258(19)41953-4
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
J. H. Harrison
中科院分区：
文献类型：
--
作者：
M. Wimmer;T. Mo;D. Sawyers;J. H. Harrison
温度研究表明，从 0 到 37 度，5-磷酸吡哆醛 (pyridoxal-5-P) 对猪心脏线粒体苹果酸脱氢酶的灭活作用是双相的，呈时间依赖性。失活的初始阶段是可逆的，但可以通过硼氢化钠还原使其不可逆。还原后的吡哆醛-5-P-酶加合物在 325 nm 处激发时在 325 nm 处表现出新的吸光度最大值，并在 325 nm 处激发时在 392 nm 处发出荧光。失活的不可逆第二阶段伴随着在没有还原的情况下出现新的 325 nm 吸光度最大值，并且在 325 处激发时荧光发射集中在约 390 至 400 nm 处。所提供的证据表明希夫的形成在失活的第一阶段，吡哆醛-5-P和苹果酸脱氢酶的亲核残基（很可能是赖氨酸）之间存在碱基。 X-唑烷样结构是席夫碱的进一步衍生物，其光谱特性与报道的数据一致，可能在第二阶段形成；这可能涉及酶的第二个亲核残基，暗示在此情况下吡哆醛-5-P作为双功能试剂的作用。辅酶 NADH 的存在可以保护该酶免于失活，这表明吡哆醛-5-P 在苹果酸脱氢酶活性中心处或附近相互作用。使用吡哆醛-5-P 与已知的苹果酸脱氢酶竞争性抑制剂 AMP、ADP 和烟酰胺进行的同时结合研究表明，吡哆醛-5-P 修饰发生在辅酶结合位点的 ADP 部分的一般区域。此外，烟酰胺的存在增强了pyridoxal-5-P与苹果酸脱氢酶的结合并使其失活。
Temperature studies have indicated that from 0 to 37 degrees, the time-dependent inactivation of mitochondrial malate dehydrogenase from porcine heart by pyridoxal 5-phosphate (pyridoxal-5-P) is biphasic. The initial phase of the inactivation is reversible but can be made irreversible by reduction with sodium borohydride. The reduced pryidoxal-5-P-enzyme adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325. The irreversible second phase of the inactivation is accompanied by the appearance of a new 325-nm absorbance maximum, in the absence of reduction, and a fluorescence emission centered about 390 to 400 nm when excited at 325. The evidence presented suggests the formation of a Schiff base between pyridoxal-5-P and a nucleophilic residue, most likely lysine, of malate dehydrogenase during the first phase of inactivation. An X-azolidine-like structure, a further derivative of the Schiff base, possessing spectral properties consistent with the reported data, may be formed during the second phase; this presumably involves a second nucleophilic residue of the enzyme, implicating the action of pyridoxal-5-P as a bifunctional reagent in this instance. The presence of the coenzyme, NADH, protects the enzyme from inactivation, suggesting that pyridoxal-5-P interacts at or near the malate dehydrogenase active center. Simultaneous binding studies using pyridoxal-5-P with known malate dehydrogenase competitive inhibitors AMP, ADP, and nicotinamide indicate that the pyridoxal-5-P modification occurs in the general area of the ADP portion of the coenzyme binging site. Furthermore, the presence of nicotinamide enhances pyridoxal-5-P binding to and inactivation of malate dehydrogenase.