Nakanishi Y, Isohashi F, Ebisuno S, Sakamoto Y
Department of Oncology, Osaka University Medical School, Japan.
Biochemistry. 1988 Jun 28;27(13):4822-6. doi: 10.1021/bi00413a036.
Cold labile extramitochondrial acetyl-CoA hydrolase (dimeric form) purified from rat liver was activated by various nucleoside triphosphates and inhibited by various nucleoside diphosphates. Activation of acetyl-CoA hydrolase by ATP was inhibited by a low concentration of ADP (Ki congruent to 6.8 microM) or a high concentration of AMP (Ki congruent to 2.3 mM). ADP and AMP were competitive inhibitors of ATP. A Scatchard plot of the binding of ATP to acetyl-CoA hydrolase (dimer) at room temperature gave a value of 25 microM for the dissociation constant with at least 2 binding sites/mol of dimer. Cold-treated monomeric enzyme also associated with ATP-agarose, suggesting that the monomeric form of the enzyme also has a nucleotide binding site(s), probably at least 1 binding site/mol of monomer. Phenylglyoxal or 2,3-butanedione, both of which modify arginyl residues of protein, inactivated acetyl-CoA hydrolase. ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by these reagents, while ADP (an inhibitor) greatly (a substratelike, competitive inhibitor), and CoASH (a product) were less effective. However, addition of ADP plus valeryl-CoA (or CoASH) effectively prevented the inactivation by 2,3-butanedione, but that is not the case for phenylglyoxal. These results suggest that one or more arginyl residues are involved in the nucleotide binding site of extramitochondrial acetyl-CoA hydrolase and that their nucleotide binding sites locate near the substrate binding site.
从大鼠肝脏中纯化得到的冷不稳定型线粒体外乙酰辅酶A水解酶（二聚体形式）可被多种核苷三磷酸激活，并被多种核苷二磷酸抑制。ATP对乙酰辅酶A水解酶的激活作用可被低浓度的ADP（Ki约为6.8微摩尔）或高浓度的AMP（Ki约为2.3毫摩尔）抑制。ADP和AMP是ATP的竞争性抑制剂。在室温下，ATP与乙酰辅酶A水解酶（二聚体）结合的Scatchard图给出的解离常数为25微摩尔，每摩尔二聚体至少有2个结合位点。经冷处理的单体酶也能与ATP琼脂糖结合，这表明该酶的单体形式也有核苷酸结合位点，可能每摩尔单体至少有1个结合位点。苯乙二醛或2,3 -丁二酮均可修饰蛋白质的精氨酸残基，使乙酰辅酶A水解酶失活。ATP（一种激活剂）能极大地保护乙酰辅酶A水解酶不被这些试剂失活，而ADP（一种抑制剂）则能极大地（一种类似底物的竞争性抑制剂），且辅酶A（一种产物）的效果较差。然而，添加ADP加戊酰辅酶A（或辅酶A）可有效防止2,3 -丁二酮导致的失活，但苯乙二醛则不然。这些结果表明，一个或多个精氨酸残基参与了线粒体外乙酰辅酶A水解酶的核苷酸结合位点，且它们的核苷酸结合位点位于底物结合位点附近。
