THE ROLE OF TYROSINE-121 IN COFACTOR BINDING OF 5-AMINOLEVULINATE SYNTHASE

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the he me biosynthesis in nonplant eukaryotes and some prokaryotes. It functi ons 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 Escher ichia coli aspartate aminotransferase, which has been shown to interac t 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 erythr oid 5-aminolevulinate synthase was substituted by Phe and His using si te-directed mutagenesis. The Y121F mutant retained 36% of the wild-typ e 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-typ e activity and the K-m value for glycine increased fivefold. The pK(al pha 1) values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121E and Y121H, resp ectively. The UV-visible and CD spectra of Y121F and Y121H mutants wer e similar to those of the wild-type with the exception of an absorptio n maximum shift (420 --> 395 nm) for the Y121F mutant in the visible s pectrum region, suggesting that the cofactor binds the Y121F mutant en zyme in a more unrestrained manner. Y121F and Y121H mutant enzymes als o exhibited lower affinity than the wild-type for the cofactor, reflec ted in the K-d values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 mu M for Y121F, Y121H, and the wild-type, respectively). Further, Y12 1F and Y121H proved less thermostable than the wild type. Taken togeth er, these findings indicate that Tyr-121 plays a critical role in cofa ctor binding of murine erythroid 5-aminolevulinate synthase.