Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase.
نویسندگان
چکیده
Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.
منابع مشابه
1 Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase
متن کامل
Crystal Structure of Mouse Carnitine Octanoyltransferase and Molecular Determinants of Substrate Selectivity*□S
Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, mediumor long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, w...
متن کاملSome kinetic studies on the mechanism of action of carnitine acetyltransferase.
1. Michaelis constants for substrates of carnitine acetyltransferase have been shown to be independent of the concentration of second substrate present. This applies to the forward reaction between acetyl-l-carnitine and CoASH, and to the back reaction between l-carnitine and acetyl-CoA. 2. Product inhibition of both forward and back reactions has been studied. Evidence has been obtained for in...
متن کاملThe substrate specificity of carnitine acetyltransferase.
1. A study of the acyl group specificity of the carnitine acetyltransferase reaction [acyl-(-)carnitine+CoASH right harpoon over left harpoon (-)-carnitine+acyl-CoA] has been made with the enzyme from pigeon breast muscle. Acyl groups containing up to 10 carbon atoms are transferred and detailed kinetic investigations with a range of acyl-CoA and acylcarnitine substrates are reported. 2. Acyl-C...
متن کاملThe effects of substrates on the optical rotatory dipersion of carnitine acetyltransferase.
1. The optical rotatory dispersion of carnitine acetyltransferase is altered in the presence of l-carnitine or acetyl-l-carnitine. These changes, which include an increase in the reduced mean residue rotation at 233nm. ([M'](233)), suggest that substrate binding causes the enzyme to unfold. 2. CoA and acetyl-CoA have no immediate effect on [M'](233) and CoA has no effect on the change in this p...
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عنوان ژورنال:
- The Journal of biological chemistry
دوره 279 30 شماره
صفحات -
تاریخ انتشار 2004