THE MIXED OXIDASE/DEHYDROGENASE ACTIVITIES OF HUMAN LONG-CHAIN ACYL-COA DEHYDROGENASE (LCAD)

Yuxun Zhang, PhD1; Megan E. Beck, PhD1; and Eric S. Goetzman, PhD1*

1Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA

*Corresponding author: eric.goetzman@chp.edu, 412-692-7952

The first step of mitochondrial fatty acid β-oxidation (FAO) is conducted by a family of acyl-CoA dehydrogenase flavoproteins. Three of these, long-chain acyl-CoA dehydrogenase (LCAD), very long- chain acyl-CoA dehydrogenase (VLCAD), and acyl-CoA dehydrogenase-9 (ACAD9) have largely overlapping substrate specificities in vitro, catalyzing the dehydrogenation of long-chain acyl-CoA species between 12 and 20 carbons in length. Determining the relative roles of these enzymes in human metabolism is important for understanding the physiology of FAO and the pathophysiology of genetic disorders thereof. VLCAD is the dominant long-chain enzyme in human heart and muscle, and ACAD9 has a moonlighting role as an assembly factor for Complex I of the respiratory chain.

LCAD, in contrast, is poorly understood. LCAD is expressed in human tissues not normally thought to rely upon FAO for energy such as the lung, thyroid, breast, and prostate. In mice LCAD is widely expressed and appears to fulfill the role that VLCAD does in humans. These differences suggest that human LCAD may have alternative functions.

In the present studies, we observed that recombinant human LCAD has mixed dehydrogenase/oxidase activities. In the absence of the physiological electron acceptor electron transferring flavoprotein (ETF), LCAD directly reduced oxygen to hydrogen peroxide (H2O2). Human LCAD’s oxidase activity was 12-to-25-fold greater than that of recombinant human VLCAD depending upon the substrate used (palmitoyl versus stearoyl-CoA), albeit still 35-fold lower than the activity of the peroxisomal long-chain acyl-CoA oxidase-1 (ACOX1), a pure oxidase that will not pass electrons to ETF. Recombinant ACAD9, in comparison, had negligible oxidase activity and was not further studied.

When recombinant human LCAD and VLCAD were mixed in the oxidase assay at a ratio approximating that which we observed in human liver, using palmitoyl-CoA as substrate, the oxidase activity of LCAD was suppressed. This suggested that VLCAD has a higher affinity for acyl-CoA substrate than LCAD. This was confirmed by anaerobic substrate titrations with the two enzymes mixed at various ratios.

Finally, we compared recombinant human LCAD to recombinant mouse LCAD and observed that the human enzyme has several-fold lower activity as a dehydrogenase but several-fold higher activity as an oxidase. Our data suggest that in human tissues where LCAD expression dominates over VLCAD, such as lung, thyroid, and prostate, the oxidase activity of LCAD could conceivably be a considerable source of H2O2. In human liver, where our data show that LCAD and VLCAD are co-expressed, VLCAD would be expected to outcompete LCAD for acyl-CoA substrate and H2O2 generation would be low. However, rising acyl-CoA concentrations or insufficient VLCAD activity (i.e., VLCAD deficiency) could result in elevated H2O2 production. Lastly, our data

showing higher oxidase/lower dehydrogenase activity for human LCAD compared to the mouse enzyme helps to explain observed inter-species differences with regards to the physiological role of LCAD.