Carboxylation of beta-methylcrotonyl coenzyme A by a purified enzyme from chicken liver.

In an earlier s tudy 1, 2 of leucine metabolism in crude heart extracts, da ta were obtained indicating that fl-methylcrotonyl CoA is acted upon by enoyl hydrase (crotonase) and tha t the resulting fl-hydroxyisovaleryl CoA is carboxylated enzymically in the presence of ATP to yield fl-hydroxy-fl-methylglutaryl CoA. This product is known 8 to be a t tacked by a specific cleavage enzyme to finnish equimolar amounts of acetoacetate and acetyl CoA. LYNEN AND KNAPPE 4-6 have recently made the interesting discovery, however, tha t fl-methylcrotonyl CoA is carboxylated directly in crotonase-free enzyme preparations from Mycobacterium spp. fl-Methylglutaconyl CoA, the product of this carboxylation reaction, is hydra ted to yield fl-hydkoxy-fl-methylglutaryl CoA by the action of methylglutaconase, a specific hydrase present in microorganisms and in animal tissues 7. During the past two years we have been engaged in the purification of the relatively unstable carboxylase from chicken liver in the hope tha t interfering enzymes could be removed and the nature of this reaction in animal tissues could be established with finality. Wi th a Ioo-fold purified preparation of the carboxylase, a requirement for methylglutaconase in the overall conversion of f l-methylcrotonyl CoA to acetoacetate has been established (Table I). Furthermoie, by employing carboxylase and cleavage-enzyme preparations preincubated with p-chloromercuribenzoate to inhibit the residual crotonase, fl-methylcrotonyl CoA, rather than r: hydroxyisovaleryl CoA, has been identified as the true substrate for the chicken-liver carboxylase (Table II) . The further unexpected finding has been made that fl-methylvinylacetyl CoA also gives rise to acetoacetate in this carboxylase system in the absence of added crotonase. The absorption maximum characteristic of a,fl-unsaturated thiol esters (~t = 267 m~ for f l-methylcmtonyl CoA) does not appear when