1. Two mitochondrial forms of acetoacetyl-CoA thiolases designated as enzyme A and enzyme B were crystallized from ox liver. They could be shown to be homogenous by polyacrylamide gel electrophoresis. 2. In direction of acetoacetyl-CoA cleavage enzyme A shows a double competitive substrate inhibition when acetoacetyl-CoA is varied at different fixed CoA concentrations. With enzyme B a parallel kinetic pattern is obtained when acetoacetyl-CoA is varied at different fixed CoA concentrations. In direction of acetoacetyl-CoA synthesis both enzymes show linear reciprocal plots of initial velocities against acetyl-CoA concentrations in absence of CoA. These initial velocity kinetics in the forward and in the reverse direction are in accordance with a ping-pong mechanism of reaction for both enzymes involving an acetyl-S-enzyme as intermediate. 3. Under saturating concentrations of substrate, the ratios of acetoacetyl-CoA synthesis/aceto-acetyl-CoA cleavage is 0.31 for enzyme A and 0.08 for enzyme B. The maximum velocity in direction of acetoacetyl-CoA synthesis of enzymes A and B are 0.43 mumol X min-1 X unit thiolase-1 and 0.10 mumol X min-1 X unit thiolase-1, respectively. 4. Both enzymes show nearly the same affinity for acetyl-CoA. The Km values are 91 muM (enzyme A) and 80 muM (enzyme B). 5. Coenzyme A and acetoacetyl-CoA both act as inhibitors in direction of acetoacetyl-CoA synthesis: coenzyme A is a nonlinear competitive inhibitor of both enzymes. Acetoacetyl-CoA exerts a negative cooperativity on enzyme A (nH = 0.63) and is a competitive inhibitor for enzyme B (Ki = 1.6 muM). 6. The catalytic and regulatory properties of the acetoacetyl-CoA thiolases A and B are discussed in terms of their proposed role in regulating ketogenesis. Intracellular fluctuations of acetoacetyl-CoA/3-hydroxybutyryl-CoA ratios, resulting in a suspension of inhibition of both enzymes at high NADH/NAD ratios, are postulated as a control mechanism of ketogenesis in addition to mechanisms already known.

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http://dx.doi.org/10.1111/j.1432-1033.1975.tb02476.xDOI Listing

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