3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties.
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 mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-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 two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.
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D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D000214:Acyl Coenzyme A; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000302:isolation & purification* / Q000378:metabolism; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D014461:Ultracentrifugation
D000214:Acyl Coenzyme A; D000596:Amino Acids; C010667:acetoacetyl CoA; D000105:Acetyl Coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase; D007652:Oxo-Acid-Lyases