Loewenthal R, Sancho J, Reinikainen T, Fersht A R
MRC Unit for Protein Function and Design, Cambridge, England.
J Mol Biol. 1993 Jul 20;232(2):574-83. doi: 10.1006/jmbi.1993.1412.
Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat/Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1.0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0.3-0.5 kcal mol-1 at a distance of 12 A, and fall gradually with distance to 0.05-0.3 kcal mol-1 at 20 A. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.
已测定了巴那斯酶和枯草杆菌蛋白酶中表面电荷之间的长程库仑相互作用能，以提供校准理论方法的数据。通过滴定色氨酸94的荧光可以准确测量巴那斯酶中组氨酸18的pKa，色氨酸94的荧光在组氨酸18质子化时会显著淬灭。枯草杆菌蛋白酶的活性位点碱基组氨酸64的pKa先前已表明可根据底物水解的kcat/Km对pH的依赖性准确测量。两个组氨酸残基的滴定曲线都非常精确地符合单基团电离的理论方程；野生型和突变型酶的希尔常数都接近1.0。通过这些带电荷侧链突变时这些残基pKa的变化，测量了远处带电荷侧链与组氨酸18和组氨酸64质子化形式之间的库仑相互作用能。在低离子强度下，蛋白质表面单个电荷之间的相互作用能很小，在12 Å距离处约为0.3 - 0.5 kcal mol-1，并随着距离逐渐下降，在20 Å处为0.05 - 0.3 kcal mol-1。多个突变通常具有加和性。枯草杆菌蛋白酶中的效应比巴那斯酶中的更大，这可能与电荷的溶剂暴露程度有关。这些数据已用于对程序DelPhi中实现的计算静电相互作用的有限差分法进行基准测试。计算结果与测量结果在位置和离子强度方面都有合理的一致性。
