tailieunhanh - Báo cáo khoa học: Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD(P) + -dependent malic enzyme Functional roles of the nucleotide binding site Lys346

Human mitochondrial NAD(P) + -dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleo-tide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. | ỊFEBS Journal Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD P -dependent malic enzyme Functional roles of the nucleotide binding site Lys346 Ju-Yi Hsieh1 Guang-Yaw Liu2 and Hui-Chih Hung1 3 1 Department of Life Sciences NationalChung-Hsing University Taichung Taiwan 2 Institute of Immunology Chung-Shan MedicalUniversity Taichung Taiwan 3 Institute of Bioinformatics NationalChung-Hsing University Taichung Taiwan Keywords allosteric regulation ATP inhibition cofactor specificity cooperativity mutagenesis Correspondence . Hung Department of Life Sciences and Institute of Bioinformatics National Chung-Hsing University 250 Kuo-Kuang Road Taichung 40227 Taiwan Fax 886 4 22851856 Tel 886 4 22840416 ext. 615 E-mail hchung@ Received 21 June 2008 revised 1 September 2008 accepted 4 September 2008 doi Human mitochondrial NAD P -dependent malic enzyme m-NAD-ME is a malic enzyme isoform with dual cofactor specificity ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms we have examined the possible role of Lys346 by replacing it to alanine serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD to NADP upon increasing kcat NADP and decreasing Km NADP. Furthermore the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wildtype is close to 2 but those of the K346 mutants are approximately . The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate suggesting