The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

Abstract

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg(2+) inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg(2+) but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a K(i) of 67mum. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

On the Mechanism of Ketogenesis and Its Control

“…They are rather a consequence of a competition bctween acetoacetyl-CoA and CoA for the free form of enzyme A and B. CoA reacts both as substrate in the normal reaction sequence and with the free enzyme. Similar results are also obtained in studies with the cytoplasmic acetoacetyl-CoA thiolase [12].…”

“…10 and 11) are consistent with the formation of a stable enzyme form F according to Cleland's notation [13] and exclude a sequential mechanism. This stable form is identical with acetyl-S-enzyme as already demonstrated for pig heart acetoacetyl-CoA thiolase and the cytoplasmic acetoacetyl-CoA thiolase from rat liver [21,12]. If there are reversible connections between the points of combination of acetyl-CoA, reciprocal plots of initial velocities versus substrate concentrations should become parabolic [13].…”

Regulation of Ketogenesis

“…The kinetic experiments were exclusively performed with transferase B, first with potassium, an activator [31], and then with sodium present. This enzyme form is not modified by bound CoASH, shows in vitro no charge heterogeneity, exhibits the higher specific activity and therefore has to be looked upon as the more active, native acetyl-CoA acetyltransferase [34].…”

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

“…Asterisks indicate the occurrence of substrate inhibition. reported before by others for cTh [1,3]. These similarities in chromatographic behaviour as well as the low activity of pTh in whole peroxisomes in comparison to the activities of the known peroxisomal thiolases suggested that we might have purified a contaminant cytosolic acetoacetyl-CoA thiolase from the peroxisomal fraction.…”