Site-Specific 13 C-Labeling of Trp 62 in Hen Egg-White Lysozyme: Preparation and ¹³C-NMR Titration of [δ1-13C] Trp 62-Lysozyme

Abstract

The indole C-2(δ1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with ^l3C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K¹³CN, NaBH₄-reduction, and acid-catalyzed dehydration. [δ1-¹³C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single ¹³C-NMR signal at 125 ppm. pH-titration of the ¹³C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GleNAc)₃, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pK_as of 3.9 and 6.5, respectively. In [2-¹³C] AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H- [2-¹³C]indo1-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differ-ently from Trp 62 on pH-titration of the ¹³C-label. These results suggest that a confor-mational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.