Serine 254 Enhances an Induced Fit Mechanism in Murine 5-Aminolevulinate Synthase

Abstract

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 Å of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K m SCoA and k cat values of the S254A variant increased, by 25-and 2-fold, respectively, the S254T substitution decreased k cat without altering K m SCoA . Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ϳ3-fold, whereas that of S254T diminished ϳ3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire ␣-oxoamine synthase family of PLP-dependent enzymes.

Toward Heme: 5-Aminolevulinate Synthase and Initiation of Porphyrin Synthesis

“…Finally, the off-rate constant determined from quenching the intrinsic ALAS fluorescence on ALA binding also matched the k cat value. 95 Overall, these findings bolstered the model originally proposed by Hunter and Ferreira 93 that the ALAS-catalyzed reaction rate is not simply limited by the dissociation of ALA from the enzyme but rather by a conformational change associated with its dissociation 74,95,97,98 (see Section XI-E).…”

“…85 Since the repositioning of Ser-189 during the ALAS conformational transition occurs in concert with the binding of succinyl-CoA, Lendrihas et al 95 proposed that this serine might couple the shift in conformational equilibrium to catalysis. The potential catalytic significance of this serine was further enhanced by the fact that this amino acid is not only perfectly conserved in all known ALAS sequences but also is conserved in all enzymes of the α-oxoamine synthase family.…”

“…[89][90][91] The ability to purify recombinant ALAS in sufficiently large quantities enabled detailed investigations of the structural and kinetic properties of the enzyme. [92][93][94][95][96][97] In fact, the understanding of the catalytic role of individual active site amino acid was largely dependent on the ability to purify murine ALAS2 in sufficiently large quantities required for detailed examinations of the biochemical and biophysical properties of the enzyme variants. 74 Moreover, elucidation of the kinetic mechanism of ALAS would have been incomplete without the ability to successfully overexpress the recombinant enzyme, considering the significant quantities of protein required for transient kinetic analyses.…”

“…The potential catalytic significance of this serine was further enhanced by the fact that this amino acid is not only perfectly conserved in all known ALAS sequences but also is conserved in all enzymes of the α-oxoamine synthase family. 95 To define the role of the equivalent murine ALAS2 serine (S254) in catalysis, Lendrihas et al 95 replaced S254 with either alanine or threonine and examined the spectroscopic and kinetic properties of the S254A and S254T variants in relation to those of the wild-type enzyme. With the serine to alanine mutation, the side chain hydroxyl group and the possible hydrogen bond with the PLP cofactor would be eliminated, while the addition of a methyl group with the threonine substitution would retain the possibility for hydrogen bonding with the cofactor and substrate, albeit steric hindrance could not be ruled out in this region of the active site.…”

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

“…This temperature was chosen for initial characterization of the steady-state kinetic properties because the majority of our studies on wild-type mALAS2 and mALAS2 variants involving enzymatic activity determinations have been conducted at 30°C (29,34,35). Relative to the wild-type enzyme, the most pronounced reduction in the k cat value was observed with the N150H and N150F variants, although the N150W mutation also reduced the turnover rate by about 40% ( Table 1).…”