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The role of tyrosine 121 in cofactor binding of 5‐aminolevulinate synthase
Author(s) -
Tan Dongwei,
Barber Michael J.,
Ferreira Gloria C.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070516
Subject(s) - cofactor , mutant , enzyme , biochemistry , tyrosine , wild type , pyridoxal , chemistry , atp synthase , biosynthesis , pyridoxal phosphate , site directed mutagenesis , heme , biology , gene
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 K m value for substrate glycine increased 34‐fold, while the activity of the Y121H mutant decreased to 5% of the wild‐type activity and the K m value for glycine increased fivefold. The p K al 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 K d values for pyridoxal 5′‐phosphate (26.5, 6.75, and 1.78 μ. 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.