Unstable Reaction Intermediates and Hysteresis during the Catalytic Cycle of 5-Aminolevulinate Synthase

Background: Aminolevulinate synthase (ALAS) catalyzes decarboxylative condensation of glycine with succinyl-CoA yielding 5-aminolevulinate. Results: Unstable ALAS-catalyzed reaction intermediates and conformational changes were characterized using physiological and non-physiological substrates and promiscuous T148A variant. Conclusion: Rate of ALA release is controlled by a hysteretic kinetic mechanism initiated by ALAS conformational changes. Significance: Unraveling the ALAS catalytic pathway enhances possible development of therapies for heme synthesis-associated disorders. 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.

• Publication year

  2014

• Venue

  Journal of Biological Chemistry

• Publication date

  2014-06-11

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.M114.574731PMID 24920668PMCID PMC4132793
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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