Effect of malonyl-CoA on the kinetics and substrate cooperativity of membrane-bound carnitine palmitoyltransferase of rat heart mitochondria.
The effect of malonyl-CoA on the kinetic parameters of carnitine palmitoyltransferase (outer) the outer form of carnitine palmitoyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) from rat heart mitochondria was investigated using a kinetic analyzer in the absence of bovine serum albumin with non-swelling conditions and decanoyl-CoA as the cosubstrate. The K0.5 for decanoyl-CoA is 3 microM for heart mitochondria from both fed and fasted rats. Membrane-bound carnitine palmitoyltransferase (outer) shows substrate cooperativity for both carnitine and acyl-CoA, similar to that exhibited by the enzyme purified from bovine heart mitochondria. The Hill coefficient for decanoyl-CoA varied from 1.5 to 2.0, depending on the method of assay and the preparation of mitochondria. Malonyl-CoA increased the K0.5 for decanoyl-CoA with no apparent increase in sigmoidicity or Vmax. With 20 microM malonyl-CoA and a Hill coefficient of n = 2.1, the K0.5 for decanoyl-CoA increased to 185 microM. Carnitine palmitoyltransferase (outer) from fed rats had an apparent Ki for malonyl-CoA of 0.3 microM, while that from 48-h-fasted rats was 2.5 microM. The kinetics with L-carnitine were variable: for different preparations of mitochondria, the K0.5 ranged from 0.2 to 0.7 mM and the Hill coefficient varied from 1.2 to 1.8. When an isotope forward assay was used to determine the effect of malonyl-CoA on carnitine palmitoyltransferase (outer) activity of heart mitochondria from fed and fasted animals, the difference was much less than that obtained using a continuous rate assay. Carnitine palmitoyltransferase (outer) was less sensitive to malonyl-CoA at low compared to high carnitine concentrations, particularly with mitochondria from fasted animals. The data show that carnitine palmitoyltransferase (outer) exhibits substrate cooperativity for both acyl-CoA and L-carnitine in its native state. The data show that membrane-bound carnitine palmitoyltransferase (outer) like carnitine palmitoyltransferase purified from heart mitochondria exhibits substrate cooperativity indicative of allosteric enzymes and indicate that malonyl-CoA acts like a negative allosteric modifier by shifting the acyl-CoA saturation to the right. A slow form of membrane-bound carnitine palmitoyltransferase (outer) was not detected, and thus, like purified carnitine palmitoyltransferase, substrate-induced hysteretic behavior is not the cause of the positive substrate cooperativity.
Department of Biochemistry, Michigan State University, East Lansing 48824.
C J
CJ
Fiol
J
J
Kerner
L L
LL
Bieber
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002331:Carnitine / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D005215:Fasting; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D008566:Membranes / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008933:Mitochondrial Swelling / Q000187:drug effects; D051381:Rats; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; C045561:decanoyl-coenzyme A; D008316:Malonyl Coenzyme A; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine