title: Fluorescence titrations of residue 59 and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1.
abstract: Fluorescence titrations of kynurenine and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1 were carried out by monitoring protein fluorescence through a pH change from 1.5 to 10.5. In the titration of kynurenine fluorescence at 455 nm, a few small but distinct quenching events occurred between pH 3.5 and 9.5. Three ionizable groups were found to be responsible for the individual steps of quenching observed. These groups are Glu 58 with pKa 4.6, His 40 or 92 with pKa 7.8 and Lys 41 with pKa 8.7. From this result, a subtle conformational change associated with the proton dissociation equilibria of Glu 58 and His 40 or 92 in the active site of Kyn 59-RNase T1 is suggested. The pH-titration behavior of tyrosine fluorescence in Kyn 59-RNase T1 was different from that of kynurenine fluorescence. Two acidic groups with pKa's 3.2 and 6.5 were detected as perturbants. In NFK 59-RNase T1, both N'-formylkynurenine and tyrosine showed almost the same fluorescence behavior during titration, which was characterized by two transitions between pH 3 and 8 in each titration curve. Two ionizable groups with pKa's 3.7-3.8 and 6.7-6.8 were determined. The role of the latter ionizable group is discussed in relation to the enzyme function of RNase T1. From the close similarity in structure and function between Kyn 59-RNase T1 and RNase T1, it is suggested that the same mechanism of conformational change linked to the ionization states of Glu 58 and His 40 or 92 exists in the native protein too.
mesh_terms: D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014443:Tyrosine
chemical_list: C007772:N'-formylkynurenine; D007737:Kynurenine; D014443:Tyrosine; D012260:Ribonucleases; D006163:Ribonuclease T1