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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)
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