UV resonance Raman studies on the activation mechanism of human hematopoietic prostaglandin D(2) synthase by a divalent cation, Mg(2+).
The Mg(2+) ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D(2) synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg(2+) to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615cm(-1) to 1600cm(-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 Mg(2+). Upon subsequent addition of glutathione (GSH), the Mg(2+)/H-PGDS solution showed the Tyr8 Raman band shifted to 1611cm(-1), which is 11cm(-1) higher than the frequency of the Mg(2+) complex of H-PGDS, but 4cm(-1) lower than the Mg(2+) free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg(2+) 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 pK(a) and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue.
Uchida
Yoshiko
Y
Institute of Applied Beam Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
Urade
Yoshihiro
Y
Mori
Seiji
S
Kohzuma
Takamitsu
T
D000818:Animals; D020134:Catalytic Domain; D004137:Dinitrochlorobenzene / Q000737:chemistry; D004789:Enzyme Activation; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054834:Lipocalins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D004137:Dinitrochlorobenzene; D007202:Indicators and Reagents; D054834:Lipocalins; D011994:Recombinant Proteins; D014443:Tyrosine; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D005978:Glutathione; D008274:Magnesium