The role of tyrosine 121 in cofactor binding of XXXX synthase.

@entity35571 @entity2228 @entity50731 @entity19 @entity2707 @entity492 @entity815 @entity8454 @entity526 @entity1446
@entity8454 synthase (EC 2.3.1.37) is the first enzyme in the @entity1446 biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires @entity2707 as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of @entity8454 synthases. Further, it corresponds to Tyr-70 of @entity526 @entity492 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 @entity19 erythroid @entity8454 synthase, Tyr-121 of @entity19 erythroid @entity8454 synthase was substituted by @entity815 and His using site-directed mutagenesis. The @entity35571 mutant retained 36% of the wild-type activity and the Km value for substrate @entity2228 increased 34-fold, while the activity of the @entity50731 mutant decreased to 5% of the wild-type activity and the Km value for @entity2228 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, @entity35571 , and @entity50731 , respectively. The UV-visible and CD spectra of @entity35571 and @entity50731 mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the @entity35571 mutant in the visible spectrum region, suggesting that the cofactor binds the @entity35571 mutant enzyme in a more unrestrained manner. @entity35571 and @entity50731 mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for @entity2707 (26.5, 6.75, and 1.78 microM for @entity35571 , @entity50731 , and the wild-type, respectively). Further, @entity35571 and @entity50731 proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of @entity19 erythroid @entity8454 synthase.
