Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase.
5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 A of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K(m)(SCoA) and k(cat) values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased k(cat) without altering K(m)(SCoA). Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved approximately 3-fold, whereas that of S254T diminished approximately 3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire alpha-oxoamine synthase family of PLP-dependent enzymes.
Lendrihas
Thomas
T
Department of Molecular Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Hunter
Gregory A
GA
Ferreira
Gloria C
GC
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism; D012694:Serine / Q000737:chemistry*
D012694:Serine; D000622:Aminolevulinic Acid; D000624:5-Aminolevulinate Synthetase; D010100:Oxygen