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5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the first, and regulatory, step of the heme biosynthetic pathway in nonplant eukaryotes and some bacteria. 5-Aminolevulinate synthase is a dimeric protein having an ordered kinetic mechanism with glycine binding before succinyl-CoA and with aminolevulinate release after CoA and carbon dioxide. Rapid scanning stopped-flow absorption spectrophotometry in conjunction with multiple turnover chemical quenched-flow kinetic analyses and a newly developed CoA detection method were used to examine the ALAS catalytic reaction and identify the rate-determining step. The reaction of glycine with ALAS follows a three-step kinetic process, ascribed to the formation of the Michaelis complex and the pyridoxal 5'-phosphate-glycine aldimine, followed by the abstraction of the glycine pro-R proton from the external aldimine. Significantly, the rate associated with this third step (k(3) = 0.002 s(-1)) is consistent with the rate determined for the ALAS-catalyzed removal of tritium from [2-(3)H(2)]glycine. Succinyl-CoA and acetoacetyl-CoA increased the rate of glycine proton removal approximately 250,000- and 10-fold, respectively, supporting our previous proposal that the physiological substrate, succinyl-CoA, promotes a protein conformational change, which accelerates the conversion of the external aldimine into the initial quinonoid intermediate (Hunter, G. A., and Ferreira, G. C. (1999) J. Biol. Chem. 274, 12222-12228). Rapid scanning stopped-flow and quenched-flow kinetic analyses of the ALAS reaction under single turnover conditions lend evidence for two quinonoid reaction intermediates and a model of the ALAS kinetic mechanism in which product release is at least the partially rate-limiting step. Finally, the carbonyl and carboxylate groups of 5-aminolevulinate play a major protein-interacting role by inducing a conformational change in ALAS and, thus, possibly modulating product release.

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5′-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the kcat values of the variants differed significantly (4.4 ± 0.2 min−1 vs. 21.6 ± 0.7 min−1) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.

5-Aminolevulinate synthase (ALAS) controls the rate-limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl-coenzyme A and glycine to produce 5-aminolevulinate, coenzyme-A (CoA), and carbon dioxide. ALAS is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X-ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl-CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl-CoA-binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl-CoA binding and substrate discrimination were examined using site-directed mutagenesis and a series of CoA-derivatives. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that of the wild-type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl-CoA-binding residues with hydrophobic amino acids caused a ligand-induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate-mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant-catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl-CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl-CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.

5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.

The two active sites of dimeric 5-aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are located on the subunit interface with contribution of essential amino acids from each subunit. Linking the two subunits into a single polypeptide chain dimer (2XALAS) yielded an enzyme with an approximate sevenfold greater turnover number than that of wild-type ALAS. Spectroscopic and kinetic properties of 2XALAS were investigated to explore the differences in the coenzyme structure and kinetic mechanism relative to those of wild-type ALAS that confer a more active enzyme. The absorption spectra of both ALAS and 2XALAS had maxima at 410 and 330 nm, with a greater A(410)/A(330) ratio at pH approximately 7.5 for 2XALAS. The 330 nm absorption band showed an intense fluorescence at 385 nm but not at 510 nm, indicating that the 330 nm absorption species is the substituted aldamine rather than the enolimine form of the Schiff base. The 385 nm emission intensity increased with increasing pH with a single pK of approximately 8.5 for both enzymes, and thus the 410 and 330 nm absorption species were attributed to the ketoenamine and substituted aldamine, respectively. Transient kinetic analysis of the formation and decay of the quinonoid intermediate EQ(2) indicated that, although their rates were similar in ALAS and 2XALAS, accumulation of this intermediate was greater in the 2XALAS-catalyzed reaction. Collectively, these results suggest that ketoenamine is the active form of the coenzyme and forms a more prominent coenzyme structure in 2XALAS than in ALAS at pH approximately 7.5.