Structural model of the catalytic core of carnitine palmitoyltransferase Iand 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 acylcar nitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identific ation for 200 amino acids surrounding the active site through a threading a pproach. The model is based on the three-dimensional structure of the rat e noyl-CoA hydratase, established by x-ray diffraction analysis. The study sh ows that the structural model of 200 amino acids of the catalytic site is p ractically 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), whi ch are 92/89 amino acids far from the catalytic histidine, respectively (bu t 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-f old, whereas the K-m for carnitine hardly changed. The inhibition of the mu tant CPT I by malonyl-CoA was not altered. The structural model explains th e loss of activity reported for the CPT I mutations R451A, W452A, D454G, W3 91A, del 8395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.