pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase

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Abstract
13C-NMR spectroscopy was used to estimate the pKa values for the Tyr150 (Y150) residue in wild-type and mutant class C β-lactamases. The tyrosine residues of the wild-type and mutant lactamases were replaced with 13C-labelled l-tyrosine ([phenol-4-13C]tyrosine) in order to observe the tyrosine residues selectively. Spectra of the wild-type and K67C mutant (Lys67→Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr150. Titration experiments showed that the chemical shift of the Tyr150 resonance in the wild-type enzyme is almost invariant in a range of 0.1p.p.m. up to pH11 and showed that the pKa 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 Tyr150, which is near the amino groups of Lys315 and Lys67, appears to have low solvent accessibility. These results suggest that, in the native enzyme, Tyr150 in class C β-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr150 loses a proton, a proton from Lys67 would replace it. Consequently, Tyr150 would be protonated during the entire titration.

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