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Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.

ABSTRACT: 5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the [Formula: see text] and a 97% decrease in [Formula: see text]. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the [Formula: see text] of K221V is lower than that of wild-type ALAS. For both enzymes, the [Formula: see text] value is significantly different from the [Formula: see text]. That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical [Formula: see text] and [Formula: see text] values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis.

SUBMITTER: Stojanovski BM

PROVIDER: S-EPMC4615937 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.

Stojanovski Bosko M BM Ferreira Gloria C GC

FEBS open bio 20151003

5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linke ...[more]

PMID: 26605136

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Project description:5-Aminolevulinate (ALA) synthase (ALAS), a homodimeric pyridoxal-5'-phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in metazoa, fungi and α-proteobacteria. In this review, we focus on the advances made in unraveling the mechanism of the ALAS-catalyzed reaction during the past decade. The interplay between the PLP cofactor and the protein moiety determines and modulates the multi-intermediate reaction cycle of ALAS, which involves the decarboxylative condensation of two substrates, glycine and succinyl-CoA. Substrate binding and catalysis are rapid, and product (ALA) release dominates the overall ALAS kinetic mechanism. Interconversion between a catalytically incompetent, open conformation and a catalytically competent, closed conformation is linked to ALAS catalysis. Reversion to the open conformation, coincident with ALA dissociation, defines the slowest step of the reaction cycle. These findings were further substantiated by introducing seven mutations in the16-amino acid loop that gates the active site, yielding an ALAS variant with a greatly increased rate of catalytic turnover and heightened specificity constants for both substrates. Recently, molecular dynamics (MD) simulation analysis of various dimeric ALAS forms revealed that the seven active site loop mutations caused the proteins to adopt different conformations. In particular, the emergence of a β-strand in the mutated loop, which interacted with two preexisting β-strands to form an anti-parallel three-stranded β-sheet, conferred the murine heptavariant with a more stable open conformation and prompted faster product release than wild-type mALAS2. Moreover, the dynamics of the mALAS2 active site loop anti-correlated with that of the 35 amino acid C-terminal sequence. This led us to propose that this C-terminal extension, which is absent in prokaryotic ALASs, finely tunes mammalian ALAS activity. Based on the above results, we extend our previous proposal to include that discovery of a ligand inducing the mammalian C-terminal extension to fold offers a good prospect for the development of a new drug for X-linked protoporphyria and/or other porphyrias associated with enhanced ALAS activity and/or porphyrin accumulation.

Dataset Information

Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.

Publications

Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.

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