In:
European Journal of Biochemistry, Wiley, Vol. 59, No. 2 ( 1975-11), p. 475-489
Abstract:
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. 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. Under saturating concentrations of substrate, the ratios of acetoacetyl‐CoA synthesis/acetoacetyl‐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 μmol × min −1 × unit thiolase −1 and 0.10 μmol × min −1 × unit thiolase −1 , respectively. Both enzymes show nearly the same affinity for acetyl‐CoA. The K m values are 91 μM (enzyme A) and 80 μM (enzyme B). 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 ( n H = 0.63) and is a competitive inhibitor for enzyme B ( K i = 1.6μM). 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.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1975.59.issue-2
DOI:
10.1111/j.1432-1033.1975.tb02476.x
Language:
English
Publisher:
Wiley
Publication Date:
1975
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
2172518-4
SSG:
12
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