Some kinetic studies on the mechanism of action of carnitine acetyltransferase

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Abstract
Michaelis constants for substrates of pigeon breast muscle carnitine acetyltransferase were independent of the concentration of 2nd substrate present. This applied to the forward reaction between acetyl-L-carnitine and CoASH, and to the back reaction between L-carnitine and acetyl-CoA. Product inhibition of both forward and back reactions was studied. Evidence was obtained for independent binding sites for L-carnitine and CoASH. Acetyl groups attached to either substrate occupied overlapping positions in space when the substrates were bound to the enzyme. Possible reaction mechansisms involving the ordered addition of substrates were excluded by determining kinetic constants in the presence and absence of added product. D-Carnitine and acetyl-D-carnitine inhibited competitively with respect to L-carnitine and acetyl-L-carnitine. The mechanism of action of carnitine acetyltransferase may thus involve 4 binary and 2 or more ternary enzyme complexes in rapid equilibrium with free substrates, the interconversion of the ternary complexes being the rate-limiting step. The possible intermediate formation of an acetylenzyme cannot be excluded, but this could only arise from a ternary complex.