Structural model of the catalytic core of carnitine palmitoyltransferase I and carnitine octanoyltransferase (COT): mutation of CPT I histidine 473 and alanine 381 and COT alanine 238 impairs the catalytic activity.
Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.
Morillas
M
M
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.
Gómez-Puertas
P
P
Roca
R
R
Serra
D
D
Asins
G
G
Valencia
A
A
Hegardt
F G
FG
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002333:Carnitine Acyltransferases / Q000737:chemistry* / Q000235:genetics; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D020033:Protein Isoforms; D006639:Histidine; D008316:Malonyl Coenzyme A; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase; D002334:Carnitine O-Palmitoyltransferase; D000409:Alanine