Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 May;7(5):1208–1213. doi: 10.1002/pro.5560070516

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

D Tan 1, M J Barber 1, G C Ferreira 1
PMCID: PMC2144007  PMID: 9605326

Abstract

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.

Full Text

The Full Text of this article is available as a PDF (539.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander F. W., Sandmeier E., Mehta P. K., Christen P. Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families. Eur J Biochem. 1994 Feb 1;219(3):953–960. doi: 10.1111/j.1432-1033.1994.tb18577.x. [DOI] [PubMed] [Google Scholar]
  2. Bishop D. F., Henderson A. S., Astrin K. H. Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome. Genomics. 1990 Jun;7(2):207–214. doi: 10.1016/0888-7543(90)90542-3. [DOI] [PubMed] [Google Scholar]
  3. Cox T. C., Bawden M. J., Abraham N. G., Bottomley S. S., May B. K., Baker E., Chen L. Z., Sutherland G. R. Erythroid 5-aminolevulinate synthase is located on the X chromosome. Am J Hum Genet. 1990 Jan;46(1):107–111. [PMC free article] [PubMed] [Google Scholar]
  4. Ferreira G. C., Gong J. 5-Aminolevulinate synthase and the first step of heme biosynthesis. J Bioenerg Biomembr. 1995 Apr;27(2):151–159. doi: 10.1007/BF02110030. [DOI] [PubMed] [Google Scholar]
  5. Gloss L. M., Kirsch J. F. Use of site-directed mutagenesis and alternative substrates to assign the prototropic groups important to catalysis by Escherichia coli aspartate aminotransferase. Biochemistry. 1995 Mar 28;34(12):3999–4007. doi: 10.1021/bi00012a018. [DOI] [PubMed] [Google Scholar]
  6. Gong J., Kay C. J., Barber M. J., Ferreira G. C. Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis. Biochemistry. 1996 Nov 12;35(45):14109–14117. doi: 10.1021/bi961296h. [DOI] [PubMed] [Google Scholar]
  7. Grishin N. V., Phillips M. A., Goldsmith E. J. Modeling of the spatial structure of eukaryotic ornithine decarboxylases. Protein Sci. 1995 Jul;4(7):1291–1304. doi: 10.1002/pro.5560040705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hunter G. A., Ferreira G. C. A continuous spectrophotometric assay for 5-aminolevulinate synthase that utilizes substrate cycling. Anal Biochem. 1995 Apr 10;226(2):221–224. doi: 10.1006/abio.1995.1217. [DOI] [PubMed] [Google Scholar]
  9. Kirsch J. F., Eichele G., Ford G. C., Vincent M. G., Jansonius J. N., Gehring H., Christen P. Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J Mol Biol. 1984 Apr 15;174(3):497–525. doi: 10.1016/0022-2836(84)90333-4. [DOI] [PubMed] [Google Scholar]
  10. Pan P., Jaussi R., Gehring H., Giannattasio S., Christen P. Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H. Biochemistry. 1994 Mar 15;33(10):2757–2760. doi: 10.1021/bi00176a003. [DOI] [PubMed] [Google Scholar]
  11. Riddle R. D., Yamamoto M., Engel J. D. Expression of delta-aminolevulinate synthase in avian cells: separate genes encode erythroid-specific and nonspecific isozymes. Proc Natl Acad Sci U S A. 1989 Feb;86(3):792–796. doi: 10.1073/pnas.86.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sutherland G. R., Baker E., Callen D. F., Hyland V. J., May B. K., Bawden M. J., Healy H. M., Borthwick I. A. 5-Aminolevulinate synthase is at 3p21 and thus not the primary defect in X-linked sideroblastic anemia. Am J Hum Genet. 1988 Sep;43(3):331–335. [PMC free article] [PubMed] [Google Scholar]
  14. Tan D., Ferreira G. C. Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation. Biochemistry. 1996 Jul 9;35(27):8934–8941. doi: 10.1021/bi952918m. [DOI] [PubMed] [Google Scholar]
  15. Toney M. D., Kirsch J. F. Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase. Biochemistry. 1991 Jul 30;30(30):7456–7461. doi: 10.1021/bi00244a013. [DOI] [PubMed] [Google Scholar]
  16. Toney M. D., Kirsch J. F. Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study. J Biol Chem. 1987 Sep 15;262(26):12403–12405. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES