Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR.
Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.
Meiering
E M
EM
Cambridge IRC for Protein Engineering, University Chemical Laboratory, U.K.
Bycroft
M
M
Fersht
A R
AR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D010710:Phosphates; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease