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 absor

SUBMITTER: Stojanovski BM 

PROVIDER: S-EPMC4132793 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature