Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

Eduardo López-Viñas,Assia Bentebibel,Chandrashekaran Gurunathan,Montserrat Morillas,Dolores De Arriaga,Dolors Serra,Guillermina Asins,Fausto G Hegardt,Paulino Gómez-Puertas

doi:10.1074/jbc.m700885200

Abstract

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Highlights

It has been postulated that there are two malonyl-CoA binding sites in the molecule of CPT1A [7, 8]
The removal of the segment comprised between amino acids 3 and 18 in both CPT1A and CPT1B decreased sensitivity to malonylCoA, which emphasizes the importance of the NH2 terminus before the first transmembrane region as a modulator of malonyl-CoA inhibition [17]
Several authors report data relating the inverse association between the effects of malonyl-CoA and carnitine on Carnitine palmitoyltransferase 1 (CPT1) activity. These can be summarized as follows: 1) in liver, the inhibitory effect of malonyl-CoA on CPT1 activity is weaker after starvation and ketosis, when the carnitine content of the cell increases [20, 21]; 2) tissues in which CPT1 is most sensitive to malonyl-CoA require the highest concentration of carnitine to drive the reaction [22]; 3) there is a reciprocal relationship between the affinity of carnitine and malonyl-CoA concentration, since malonylCoA increases the Km of the enzyme for carnitine, and the inhibitory effect of malonyl-CoA varies with carnitine concentration [11, 23]; 4) mutations designed to decrease malonyl-CoA sensitivity increase catalytic efficiency of CPT1 for carnitine, which appears to be roughly proportional to the extent of the alteration in malonyl-CoA sensitivity [15]

Summary

Introduction

It has been postulated that there are two malonyl-CoA binding sites in the molecule of CPT1A [7, 8]. Our group has reported that the conserved Met593, which is located in the COOH-terminal domain, plays a critical permissive role in the interaction between the enzyme and malonyl-CoA The mutation of this amino acid to serine abolished the malonyl-CoA sensitivity of CPT1A [15]. The removal of the segment comprised between amino acids 3 and 18 in both CPT1A and CPT1B decreased sensitivity to malonylCoA, which emphasizes the importance of the NH2 terminus before the first transmembrane region as a modulator of malonyl-CoA inhibition [17]. Mutations of specific amino acids from both NH2 and COOH termini support their involvement in CPT1 malonyl-CoA sensitivity This model is supported by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine, suggesting that the positioning of malonyl-CoA near to the carnitine binding locus produces competitive-noncompetitive inhibition. This conclusion is supported by experimental data and by the two putative binding sites for malonyl-CoA in the three-dimensional structural model of CPT1A deduced from docking calculations

Objectives

Results

Conclusion