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.

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.