Schreiber G, Fersht A R
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, U.K.
Biochemistry. 1993 May 18;32(19):5145-50. doi: 10.1021/bi00070a025.
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)
芽孢杆菌核糖核酸酶Barnase是解淀粉芽孢杆菌的一种细胞外核糖核酸酶，它与其细胞内多肽抑制剂芽孢杆菌抗酶形成非常紧密的复合物。在pH 8时，速率常数k1（缔合）和k-1（解离）的值分别为6.0×10⁸ s⁻¹ M⁻¹和8.0×10⁻⁶ s⁻¹。根据k-1/k1的比值计算得到的芽孢杆菌抗酶和Barnase的解离常数Ki为1.3×10⁻¹⁴ M，在25℃时对应-18.9 kcal/mol的ΔG。解离常数随着pH的降低而增加，这是由于pKa为6.4的游离Barnase中一种酸的电离，与质子化形式的结合非常弱（如果有的话）。如通过His102→Ala突变所确定的，复合物解离的这种pH依赖性几乎完全归因于Barnase中的His102残基。对动力学常数的pH依赖性分析表明，结合至少是一个两步过程。第一步也是限速步骤，是接近扩散控制速率的缔合。然后是复合物的精确对接。在500 mM NaCl存在下，Ki的值增加到2.4×10⁻¹¹ M，在pH 5（100 mM NaCl）时增加到1.6×10⁻¹¹ M。通过测量一系列广泛的Barnase位点特异性突变体的Ki值，绘制了芽孢杆菌抗酶在Barnase上的结合位点。将Lys27、Arg59、Arg87和His102残基突变为Ala会使Ki值增加10⁴倍。
