Stepp L R, Reed L J
Biochemistry. 1985 Dec 3;24(25):7187-91. doi: 10.1021/bi00346a026.
The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.
来自牛肾和心脏的丙酮酸脱氢酶多酶复合体可通过用磷酸吡哆醛和氰化钠或硼氢化钠处理而失活。这种抑制作用的位点是该复合体中的丙酮酸脱氢酶（E1）组分。硫胺素焦磷酸可防止磷酸吡哆醛 - 氰化物处理对E1的失活作用。平衡结合研究表明，E1每分子（α2β2）含有两个硫胺素焦磷酸结合位点，并且E1的修饰使硫胺素焦磷酸的解离常数（Kd）增加了约5倍。在磷酸吡哆醛存在下，每摩尔E1四聚体掺入约2.4当量的14CN导致E1活性损失约90％。放射性主要掺入E1α亚基中。在已用NaB3H4还原的E1 - 磷酸吡哆醛复合体的酸水解物中鉴定出放射性N6 - 吡啶氧基赖氨酸。这些数据被解释为表明在氰化钠或硼氢化钠存在下，磷酸吡哆醛与E1的硫胺素焦磷酸结合位点处或附近的赖氨酸残基反应。该结合位点显然位于α亚基上。
