Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR

Journal Article · Tue Nov 26 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00111a022· OSTI ID:5687178

Meiering, E M; Bycroft, M; Fersht, A R ^[1]

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1. University Chemical Lab., Cambridge (United Kingdom)

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, K{sub d}, of 1.3 mM. The 2D {sup 1}H 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 K{sub d} for phosphate by more than 20-fold. The 2D {sup 1}H NMR spectra of the mutants His-102 {yields} Ala, Lys-27 {yields} Ala, and Arg-87 {yields} Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102 {yields} Ala and Lys-27 {yields} Ala have essentially the same structure as wild type, while some structural changes occur in Arg-87 {yields} Ala. It appears that phosphate binding by barnase is effected mainly by positively charged residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.

• OSTI ID: 5687178
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:47; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID PHOSPHATES
AMIDES
BACILLUS
BACTERIA
CARBONIC ACID DERIVATIVES
CHEMICAL REACTIONS
ENZYME INDUCTION
ENZYME INHIBITORS
ENZYMES
ESTERASES
ESTERIFICATION
GENE REGULATION
HYDROLASES
MAGNETIC RESONANCE
MICROORGANISMS
MUTAGENESIS
NUCLEAR MAGNETIC RESONANCE
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHODIESTERASES
PHOSPHORUS COMPOUNDS
PROTEINS
RESONANCE
RNA-ASE
UREA