title: Unstable reaction intermediates and hysteresis during the catalytic cycle of 5-aminolevulinate synthase: implications from using pseudo and alternate substrates and a promiscuous enzyme variant.
abstract: 5-Aminolevulinate (ALA), an essential metabolite in all heme-synthesizing organisms, results from the pyridoxal 5'-phosphate (PLP)-dependent enzymatic condensation of glycine with succinyl-CoA in non-plant eukaryotes and α-proteobacteria. The predicted chemical mechanism of this ALA synthase (ALAS)-catalyzed reaction includes a short-lived glycine quinonoid intermediate and an unstable 2-amino-3-ketoadipate intermediate. Using liquid chromatography coupled with tandem mass spectrometry to analyze the products from the reaction of murine erythroid ALAS (mALAS2) with O-methylglycine and succinyl-CoA, we directly identified the chemical nature of the inherently unstable 2-amino-3-ketoadipate intermediate, which predicates the glycine quinonoid species as its precursor. With stopped-flow absorption spectroscopy, we detected and confirmed the formation of the quinonoid intermediate upon reacting glycine with ALAS. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine was reacted with ALAS, a lag phase was observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in ALAS. Hysteresis was not detected in the T148A-catalyzed L-serine external aldimine formation. These results with T148A, a mALAS2 variant, which, in contrast to wild-type mALAS2, is active with L-serine, suggest that active site Thr-148 modulates ALAS strict amino acid substrate specificity. The rate of ALA release is also controlled by a hysteretic kinetic mechanism (observed as a lag in the ALA external aldimine formation progress curve), consistent with conformational changes governing the dissociation of ALA from ALAS.
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612 and.
authors.forename: Bosko M
authors.initials: BM
authors.lastname: Stojanovski
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612 and.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Hunter
authors.affiliation: Institute of Microbiology, Technical University of Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Jahn
authors.affiliation: Institute of Microbiology, Technical University of Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612 and. Electronic address: gferreir@health.usf.edu.
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000622:Aminolevulinic Acid / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D007700:Kinetics; D051379:Mice; D020125:Mutation, Missense; D012521:Sarcosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D000622:Aminolevulinic Acid; C012046:succinyl-coenzyme A; D000624:5-Aminolevulinate Synthetase; C588337:ALAS2 protein, mouse; D012521:Sarcosine
keywords: 5-Aminolevulinate Synthase; Enzyme Kinetics; Enzyme Mechanism; Heme; Hysteresis; Oxoamine Synthase; Porphyria; Porphyrin; Pyridoxal Phosphate; Sideroblastic Anemia