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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.
Highlights
Aminolevulinate synthase (ALAS) catalyzes decarboxylative condensation of glycine with succinyl-CoA yielding 5-aminolevulinate
Samples were analyzed by full-scan Q1 positive and negative modes and multiple reaction monitoring (MRM) positive mode for the predicted ALA synthase (ALAS) intermediate (Fig. 1)
We provide direct evidence for the presence of the first quinonoid and -ketoacid reaction intermediates in the catalytic pathway of ALAS and examine the amino acid substrate specificity and conformational transitions of ALAS by focusing on the hysteretic behavior of the enzyme
Summary
Aminolevulinate synthase (ALAS) catalyzes decarboxylative condensation of glycine with succinyl-CoA yielding 5-aminolevulinate. Formation of the initial quinonoid intermediate could be spectroscopically observed upon binding of either glycine or glycine and succinyl-CoA to R. sphaeroides ALAS [20] The monitoring of this quinonoid intermediate was clearly facilitated in the Rhodobacter capsulatus ALAS-catalyzed reaction by substituting glycine with O-methylglycine [21], as the resulting methyl ester of the -ketoacid-aldimine could not undergo enzymatic decarboxylation, and accumulated. We report the direct identification of the 2-amino-3ketoadipate intermediate, (Scheme 1, V) using glycine and O-methylglycine as substrate and pseudo-substrate, respectively, and a combination of stopped-flow kinetics and liquid chromatography coupled with tandem mass spectrometry (LCMS/MS) This finding, together with the spectroscopic detection of the first quinonoid intermediate (Scheme 1, III) and consequent calculation of the rate constant associated with its formation, supports the occurrence of these two intermediates in the pathway of the ALAS-catalyzed reaction and confirms that the quinonoid intermediate (III) stems from deprotonation rather than decarboxylation of the ALAS-glycine external aldmine (Scheme 1, II). We propose that this hysteretic behavior emanates from structural rearrangements induced by the different orientation of the bound L-serine in the mALAS2 active site
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