On the Mechanism of Ketogenesis and Its Control

Abstract

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. 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 pM).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.In a systematic study of the relative labelling of the carbonyl and carboxyl group of acetoacetate formed from (1-14C)-labelled fatty acids, in 1973 Huth etal. demonstrated a variability of both groups at various metabolic states. From a preferential labelling of the carbonyl group in the diabetic state as compared to unconditioned animals showing low rates of ketogenesis, a point of regulation was located at the level

Interrelationships between 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase, Acetoacetyl‐CoA and Ketogenesis

“…For example, the recent report of McKean et al [6] suggests that acetoacetyl-CoA levels may modulate activity of acyl-CoA dehydrogenase. In addition, acetoacetyl-CoA thiolase [7, 101 have been postulated to be rate-limiting in ketogenesis.Evaluation of the role of HoMeGlt-CoA cycle enzymes in regulation of ketogenesis is difficult due to the lack of a detailed Abbrcviutions. HoMeGlt, 3-hydroxy-3-methylglutaryl; Hepes, 4-(2-hydroxyethy1)-I-piperazineethanesulfonic acid.…”

Immunological assay of the mitochondrial acetyl‐CoA acetyltransferase in crude liver homogenate

“…100 g body wt-") [12]. Obviously only a part of the maximal synthesizing activity can be expressed in vivo due to the inhibitory action of CoASH and acetaacetytCoA on the mAAT [6,7,13]. CoASH may be of physiological importance to the ketogenic rates, for the ketone body production only correlates with the The two forms of the mAAT, A and B, differ in isoelectric points and in specific activities [2].…”

Regulation of Hepatic Fatty Acid Oxidation and Ketogenesis

“…Huth et al (1975) have pointed out that the activity of the enzyme in the direction of acetoacetyl-CoA synthesis is very much lower than in the thiolytic direction, and from isotopic distribution studies they concluded that the activity of the enzyme may be rate-limiting (Huth et al I 973). One factor which could limit acetyl-CoA acetyltransferase activity in vivo is inhibition by the product of the reaction, acetoacetyl-CoA (Huth et al 1975). Acetoacetyl-CoA also inhibits 3-hydroxy-3-methyl glutaryl (HMG)-CoA synthase (Reed et al r975)-the next enzyme in the ketogenic pathway.…”

“…Since the two forms have different kinetic characteristics (Huth et al 1973) this interconversion may result in a high sensitivity of the activity of the enzyme to changes in the ratio of [acetyl-CoA]: [CoA]. Huth et al (1975) have pointed out that the activity of the enzyme in the direction of acetoacetyl-CoA synthesis is very much lower than in the thiolytic direction, and from isotopic distribution studies they concluded that the activity of the enzyme may be rate-limiting (Huth et al I 973). One factor which could limit acetyl-CoA acetyltransferase activity in vivo is inhibition by the product of the reaction, acetoacetyl-CoA (Huth et al 1975).…”