Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B₁-insensitive ceramide synthase

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B₁, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with D-erythro-sphingosine (K_m of 0.16 mol %, and V_max of 0.3 μmol/min/mg), but D-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a K_m of 1.7 mol % and a V_max of 0.63 μmol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, L-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.