Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal.
Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal.
复制标题
磷酸吡哆醛和腺苷多磷酸吡哆醛对乳酸脱氢酶的修饰。
DOI：
10.1021/bi00358a034
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
T. Fukui
中科院分区：
文献类型：
--
作者：
M. Tagaya;T. Fukui
磷酸吡哆醛不仅与酶活性必需的赖氨酰残基发生反应，而且还与兔肌肉乳酸脱氢酶 (EC 1.1.1.27) 中的其他反应性赖氨酰残基发生反应。为了提高磷酸吡哆醛的特异性，新合成了腺苷二磷酸、三磷酸和四磷酸吡哆醛并用于修饰该酶。将酶与浓度均为 1 mM 的二磷酸、三磷酸和四磷酸化合物一起孵育 30 分钟，然后用硼氢化钠还原，导致酶活性分别损失 64%、51% 和 34%。 NADH 几乎完全保护酶免于失活，而丙酮酸则没有表现出任何保护作用。等摩尔量的试剂与酶亚基的结合对应于完全失活。具有不同残留活性的腺苷二磷酸吡哆醛修饰酶在Blue Toyopearl亲和柱上进行色谱分析。结果表明，除了完整的酶之外，还存在至少四种酶，它们在试剂结合量、NADH 亲和力和比活性方面彼此显着不同。在腺苷二磷酸吡哆醛的修饰中，酶对 NADH 的亲和力的降低和酶活性的丧失是平行的，而在用磷酸吡哆醛的修饰中，对 NADH 的亲和力的降低先于失活。结论是，腺苷多磷酸吡哆醛化合物的修饰对于乳酸脱氢酶中的活性位点赖氨酰残基具有特异性。
Pyridoxal phosphate reacts with not only the lysyl residue(s) essential for enzymatic activity but also other reactive lysyl residues in rabbit muscle lactate dehydrogenase (EC 1.1.1.27). To raise the specificity of pyridoxal phosphate, adenosine diphospho-, triphospho-, and tetraphosphopyridoxals have been newly synthesized and used for modification of the enzyme. Incubation of the enzyme for 30 min with the diphospho, triphospho, and tetraphospho compounds all at 1 mM followed by reduction by sodium borohydride resulted in the loss of enzymatic activity by 64, 51, and 34%, respectively. NADH almost completely protected the enzyme from inactivation, whereas pyruvate showed no protection. Binding of the reagents to the enzyme subunit in an equimolar amount corresponds to the complete inactivation. The adenosine diphosphopyridoxal modified enzymes with different residual activities were chromatographed on a Blue Toyopearl affinity column. The results showed the presence of at least four enzyme species besides the intact enzyme that are significantly different from one another in the amount of the reagent bound, the affinity for NADH, and the specific activity. The decrease in the affinity of the enzyme for NADH and the loss of enzymatic activity paralleled in the modification by adenosine diphosphopyridoxal, whereas, in the modification by pyridoxal phosphate, the decrease in the affinity for NADH preceded the inactivation. It is concluded that modification by adenosine polyphosphopyridoxal compounds are specific for the active site lysyl residue(s) in lactate dehydrogenase.