Catalytically important domains of rat carnitine palmitoyltransferase II as determined by site-directed mutagenesis and chemical modification. Evidence for a critical histidine residue.
Rat carnitine palmitoyltransferase (CPT) II was expressed in Saccharomyces cerevisiae. Mitochondrial fractions prepared from the cells displayed high CPT activity and reacted with an antibody to the rat protein on immunoblots, whereas no activity or immunoreactive protein was observed in control cells. The recombinant enzyme was largely membrane associated. Treatment of the expressed protein with diethyl pyrocarbonate, a reagent that modifies histidine residues, abolished CPT activity, but this was completely restored by reversal of the modification with hydroxylamine. It is inferred that a histidine residue plays a critical role in CPT function. Expression and analysis of site-directed mutants of CPT II showed that histidine 372, as well as aspartates 376 and 464 (all conserved throughout the carnitine/choline acyltransferase family), are essential for catalytic activity. The data suggest that the mechanism by which CPT II effects transesterification between palmitoyl-CoA and carnitine possibly involves histidine 372 and one of these aspartate residues, interacting with the carnitine hydroxyl group, in a reaction analogous to that carried out by a histidine/aspartate/serine catalytic triad in certain other enzyme systems. Mutagenic analysis of a region of CPT II that is highly conserved among the carnitine and choline acyltransferases, and which is homologous to the "adenine binding loop" of citrate synthase, implies that it has no similar function in CPT II.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
N F
NF
Brown
R C
RC
Anderson
S L
SL
Caplan
D W
DW
Foster
J D
JD
McGarry
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001224:Aspartic Acid; D006639:Histidine; D002334:Carnitine O-Palmitoyltransferase; D002950:Citrate (si)-Synthase; D004047:Diethyl Pyrocarbonate