Acta Biochimica et Biophysica 7. (1972)

1972 / 2. szám - I. Alkonyi-A. Sándor: Demonstration of an Enzymatic Transfer Reaction between ß-Methyl-crotonyl-Coenzyme A and L-carnitine (Short Communication)

150 I. Alkonyi, A. Sándor: Demonstration of an Enzymatic Transfer Reaction method of Knappe et al. (1961), acetyl-CoA was prepared as described by Stadt­­man (Colowick, Kaplan, 1957). The above reaction was measured from left to right and the amount of CoA liberated was determined with Ellman’s reagent. As shown in Table 1, the hypothetic enzyme reaction indeed takes place, and at a reasonable rate. As suggested by Fritz et al., the rate of the transfer reaction was Table 1 Comparison of the enzymatic transfer reactions of acetyl-CoA and ß-methyl-crotonyl-CoA The cuvettes contained the following components in 2.3 ml final volume: 200 («moles of Tris-HCl, pH 7.8; 10 /«noies of EDTA; 200 nmoles of 5,5’-dithiobis (2-nitrobenzoic acid); 200 nmoles of DL-carnitine; 56 nmoles of acyl-CoA; carnitine-acetyl transferase as indicated. The reaction was started by the addition of enzyme and the mixture was then incubated at 25 °C for 30 min. At the end of the incubation period the change in absorbance at 412 nm was read and the calculations were based on the molar extinction coefficient: 13 600 M-1 cm-1. The reference cuvette did not contain carnitine compared with that of the transfer reaction of acetyl-CoA measured within the same period. As for the nature of the enzyme catalyzing the transfer reaction of /1-methyl-crotonyl group, at present we are only confined to speculations: 1. The broad specificity of the acetyl-transferase enzyme may also include the substrate tested in this work, but the reaction proceeds at a low speed. 2. The reaction observed by us may be attributed to the octanoyl-transferase activity described by Solberg (1971). 3. Finally, we may deal with an entirely new enzyme. In order to validate one of the above alternatives further experiments are required. References Bremer, J. (1962) J. Biol. Chem. 237 3628 Bressler, R. (1970) Compr. Biochem. 18 331 Bressler, R., Brendel, K. (1966) J. Biol. Chem. 241 4092 Chase, J. F. A., Pearson, D. J., Tubbs, P. K. (1965) Biochim. Biophys. Acta 96 162 Colowick, S. P., Kaplan, N. O. (1957) Methods in Enzymology, Vol. III. Academic Press Inc., Publishers, New York, pp. 931 — 932 Decker, K. (1959) Die aktivierte Essigsäure. Ferdinand. Enke Verlag, Stuttgart, pp. 262—270 Friedman, S., Fraenkel, G. (1955) Arch. Biochem. Biophys. 59 491 Fritz, J. B., Schultz, S. K., Srere, P. A. (1963) J. Biol. Chem. 238 2509 Knappe, J., Schlegel, H. G., Lynen, F. (1961) Biochem. Z. 335 101 Norum, K. R., Bremer, J. (1967) J. Biol. Chem. 242 407 Solberg, H. E. (1971) FEBS Letters 12 134 CoA liberated in the first 30 min, nmoles Substrate 0.009 enzyme 0.018 enzyme 0.04 enzyme units units units Acetyl-CoA 19.2 26.4 39.0 jS-methyl-crotonyl-CoA 9.6 15.0 18.4 Acta Biochimica et Biophysica Academiae Scientiarum Hungaricae 7, 1972

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