Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

Gloria C. Ferreira, Ph.D.

Keywords

X-linked sideroblastic anemia, Alpha-oxoamine synthase, Transient kinetics, Pyridoxal 5?-phosphate, Porphyria, Photodynamic therapy

Abstract

The biosynthesis of tetrapyrolles in eukaryotes and the alpha-subclass of purple photosynthetic bacteria is controlled by the pyridoxal 5?-phosphate (PLP)-dependent enzyme, 5-aminolevulinate synthase (ALAS). Aminolevulinate, the universal building block of these macromolecules, is produced together with Coenzyme A (CoA) and carbon dioxide from the condensation of glycine and succinyl-CoA. The three-dimensional structures of Rhodobacter capsulatus ALAS reveal a conserved active site serine that moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed, substrate-bound enzyme conformation, and simultaneously to within 3-4 angstroms of the thioester sulfur atom of bound succinyl-CoA. To elucidate the role(s) this residue play(s) in enzyme activity, the equivalent serine in murineerythroid ALAS was mutated to threonine or alanine.

The S254A variant was active, but both the KmSCoA and kcat values were increased, by 25- and 2-fold, respectively, suggesting the increase in turnover is independent of succinyl-CoA-binding. In contrast, substitution of S254 with threonine results in a decreased kcat, however the Km for succinyl-CoA is unaltered. Removal of the side chain hydroxyl group in the S254A variant notably changes the spectroscopic properties of the PLP cofactor and the architecture of the PLP-binding site as inferred from circular dichroism spectra. Experiments examining the rates associated with intrinsic protein fluorescence quenching of the variant enzymes in response to ALA binding show that S254 affects product dissociation. Together, the data led us to suggest that succinyl-CoA binding in concert with the hydrogen bonding state of S254 governs enzyme conformational equilibria.

As a member of the alpha-oxoamine synthase family, ALAS shares a high degree of structural similarity and reaction chemistry with the other enzymes in the group. Crystallographic studies of the R. capsulatus ALAS structure show that the alkanoate component of succinyl-CoA is bound by a conserved arginine and a threonine. To examine acyl-CoA-binding and substrate discrimination in murine erythroid ALAS, the corresponding residues (R85 and T430) were mutated and a series of CoA substrate analogs were tested. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that calculated for the wild-type enzyme, suggesting this residue is strategic in substrate binding. Hydrophobic substitutions of the residues that coordinate acyl-CoA-binding produce ligand-induced changes in the CD spectra, indicating that these amino acids affect substrate-mediated changes to the microenvironment of the chromophore.

Pre-steady-state kinetic analyses of the R85K variant-catalyzed reaction show that both the rates associated with product-binding and the parameters that define quinonoid intermediate lifetime are dependent on the chemical composition of the acyl-CoA tail. Each of the results in this study emphasizes the importance of the relationship between the bifurcate interaction of the alkanoic acid component of succinyl-CoA and the side chains of R85 and T430.

From the X-ray crystal structures of Escherichia coli 8-amino-7-oxonoanoate synthase and R. capsulatus ALAS, it was inferred that a loop covering the active site moved 3-6 Å between the holoenzymic and acyl-CoA-bound conformations. To elucidate the role that the active site lid plays in enzyme function, we shuffled the portion of the murine erythroid ALAS cDNA corresponding to the lid sequence (Y422-R439), and isolated functional variants based on genetic complementation in an ALA-deficient strain. Variants with potentially greater enzymatic activity than the wild-type enzyme were screened for increased porphyrin overproduction. Turnover number and the catalytic efficiency of selected functional variants with both substrates were increased for each of the enzyme variants tested, suggesting that increased activity is linked to alterations of the loop motif. The results of transient kinetics experiments for three isolated variants when compared to wild-type ALAS showed notable differences in the pre-steady-state rates that define the kinetic mechanism, indicating that the rate of ALA release is not rate-limiting for these enzymes. The thermodynamic parameters for a selected variant-catalyzed reaction indicated a reduction in the amount of energy required for catalysis. This finding is consistent with the proposal that, in contrast to the wild-type ALAS reaction, a protein conformational change associated with ALA release no longer limits turnover for this variant enzyme.

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