Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1316134 | Journal of Inorganic Biochemistry | 2010 | 10 Pages |
The Mg2+ ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D2 synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg2+ to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615 cm−1 to 1600 cm−1. This large shift to lower energy of the tyrosine Y8a vibrational mode is caused by the deprotonation of the tyrosine phenol group promoted by binding of Mg2+. Upon subsequent addition of glutathione (GSH), the Mg2+/H-PGDS solution showed the Tyr8 Raman band shifted to 1611 cm−1, which is 11 cm−1 higher than the frequency of the Mg2+ complex of H-PGDS, but 4 cm−1 lower than the Mg2+ free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg2+ is re-protonated by the abstraction of H+ from the thiol group of GSH, and that the re-protonated Tyr8 species forms a hydrogen bond with the thiolate anion of GSH. Density functional theory calculations on several model complexes of p-cresol were also performed, which suggested that the pKa and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue.