pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase.
13C-NMR spectroscopy was used to estimate the p K a values for the Tyr(150) (Y150) residue in wild-type and mutant class C beta-lactamases. The tyrosine residues of the wild-type and mutant lactamases were replaced with (13)C-labelled L-tyrosine ([ phenol -4-(13)C]tyrosine) in order to observe the tyrosine residues selectively. Spectra of the wild-type and K67C mutant (Lys(67)-->Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr(150). Titration experiments showed that the chemical shift of the Tyr(150) resonance in the wild-type enzyme is almost invariant in a range of 0.1 p.p.m. up to pH 11 and showed that the p K (a) of this residue is well above 11 in the substrate-free form. According to solvent accessibility calculations on X-ray-derived structures, the phenolic oxygen of Tyr(150), which is near the amino groups of Lys(315) and Lys(67), appears to have low solvent accessibility. These results suggest that, in the native enzyme, Tyr(150) in class C beta-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr(150) loses a proton, a proton from Lys(67) would replace it. Consequently, Tyr(150) would be protonated during the entire titration.
Kato-Toma
Yoko
Y
Suntory Institute for Bioorganic Research, 1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan.
Iwashita
Takashi
T
Masuda
Katsuyoshi
K
Oyama
Yoshiaki
Y
Ishiguro
Masaji
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D016971:Citrobacter freundii / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D017354:Point Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017186:Titrimetry; D014443:Tyrosine / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000145:classification / Q000235:genetics
D010446:Peptide Fragments; D014443:Tyrosine; D001618:beta-Lactamases; D008239:Lysine