Binding of nucleotides to an extramitochondrial acetyl-CoA hydrolase from rat liver

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Abstract
Cold labile extramitocondrial acetyl-CoA hydrolase (dimeric form) purified from rat liver was activated by various nucleotide trisphosphates and inhibited by various nucleotide diphosphates. Activation of acetyl-CoA hydrolase by ATP was inhibited by a low concentration of ADP (Ki .simeq. 6.8 .mu.M) or a high concentration of AMP (Ki .simeq. 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 .mu.M for the dissociation constant with at least 2 binding sites/mol or dimer. Cold-treated monomeric enzyme also associated with ATP-agarose, suggesting that the monomeric form of the enzyme is also has a nucleotide binding site(s), probably at least 1 binding site/mol of monomer. Phenylglyoxal or 2,3-butandione, 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), valeryl-CoA (a substratelike, competitive inhibitor), and CoASH (a product) were less effective. However, addition of ADP plus valeryl-CoA (or CoASH) effectively prevented the inaction 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.

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