Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering.
Barnase, an extracellular ribonuclease of Bacillus amyloliquefaciens, forms a very tight complex with its intracellular polypeptide inhibitor barstar. At pH 8, the values for the rate constants k1 (association) and k-1 (dissociation) are 6.0 x 10(8) s-1 M-1 and 8.0 x 10(-6) s-1, respectively. The value of Ki, the dissociation constant of barstar and barnase, calculated from the ratio k-1/k1 is 1.3 x 10(-14) M, which corresponds to a delta G of -18.9 kcal/mol at 25 degrees C. The dissociation constant increases with decreasing pH according to the ionization of an acid in free barnase of pKa 6.4, with very weak, if any, binding to the protonated form. This pH dependence for dissociation of the complex can be attributed almost entirely to residue His102 in barnase, as determined by a His102-->Ala mutation. Analysis of the pH dependence of the kinetic constants indicates that binding is, at least, a two-step process. The first, and rate-determining, step is association at close to the diffusion-controlled rate. There is then the precise docking of the complex. The value of Ki increases to 2.4 x 10(-11) M in the presence of 500 mM NaCl, and to 1.6 x 10(-11) M at pH 5 (100 mM NaCl). The binding site of barstar on barnase was mapped by measuring the values of Ki for a broad range of site-specific mutants of barnase. Mutagenesis of residues Lys27, Arg59, Arg87, and His102 to Ala increases the values of Ki by a factor of 10(4).(ABSTRACT TRUNCATED AT 250 WORDS)
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, U.K.
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D001120:Arginine; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D016296:Mutagenesis; D015202:Protein Engineering; D012260:Ribonucleases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001426:Bacterial Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D012965:Sodium Chloride; D006639:Histidine; D001120:Arginine; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D008239:Lysine