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Originally published In Press as doi:10.1074/jbc.M002118200 on May 8, 2000

J. Biol. Chem., Vol. 275, Issue 29, 22020-22024, July 21, 2000
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Identification by Mutagenesis of Conserved Arginine and Tryptophan Residues in Rat Liver Carnitine Palmitoyltransferase I Important for Catalytic Activity*

Jia Dai, Hongfa Zhu, Jianying ShiDagger , and Gebre Woldegiorgis§

From the Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of L-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the Km values for carnitine or palmitoyl-CoA. However, these mutations decreased the Vmax values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha -helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.

* This work was supported by National Institutes of Health Grant HL52571 (to G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Bioinformatics Group, Cereon Genomics, 45 Sidney St., Cambridge, MA 02139.

§ To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. Walker Rd., Beaverton, OR 97006-8921. Tel.: 503-748-1686; Fax: 503-748-1464; E-mail: gwoldeg@bmb.ogi.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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