PubMed · 8752976
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T Hotta. 1996. [Bone marrow failure--current progress in pathophysiology].. https://pubmed.ncbi.nlm.nih.gov/8752976/
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5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.
The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits (p45 and p18), each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an enhancer-binding protein for expression of the beta-globin gene. We show here that dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells, but not a mutant clone of dimethyl sulfoxide-resistant cells, increases NF-E2 activity significantly, which involves both up-regulation of DNA binding and transactivation activities. Both activities were reduced markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH2-terminal region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine erythroleukemia cells, and serine/threonine phosphorylation may be involved in this process. In addition, they also suggest that a MafK homodimer can suppress transcription, not only by competition for the DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.
5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme. In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.