3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties

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Abstract
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mass was about 100,000 and .**GRAPHIC**. [N.a] was 6.36S. The enzyme appears to be a dimer of identical subunits (MW 47,900). The Km for acetoacetyl-CoA is extremely low (0.5 .mu.M) and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition .**GRAPHIC**. = 3.5 .mu.M) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with 2 forms of the enzyme. The 2 forms are most likely to be free enzyme and an acetyl-enzyme intermediate.