Warwicker J, Mueller-Harvey I, Sumner I, Bhat K M
Protein Engineering Department, AFRC Institute of Food Research Reading Laboratory, U.K.
J Mol Biol. 1994 Feb 25;236(3):904-17. doi: 10.1006/jmbi.1994.1197.
The activity of porcine pancreatic phospholipase A2 (pla2), measured at pH 8, is reduced when methanol or ethanol is added to the aqueous solution. Finite difference electrostatics calculations were used to study the effect of modelling mixed solvents on the pKas of histidine 48 and the amino-terminal group, both of which influence the pH-dependence of catalysis. Calculations and experiment indicate that these pKa values cannot account for the activity reduction. Charge separation in the transition state is destabilized in 20% alcohol solvent relative to 100% aqueous solvent. The calculated values, which are combinations of stabilizing and destabilizing contributions, are in qualitative agreement with experiment. Saturating dielectric theory is used to model solvent water ordering in a high electric field, and water dielectric structure is assumed to dominate at the 20% alcohol level. The observed agreement demonstrates the utility of transition state stabilization theory and continuum solvent modelling. It is further suggested that electrostatic effects on kcat contribute to the pH-dependence of activity around pH 7, and to previously reported activity changes for charge mutants.
在pH值为8时测定的猪胰磷脂酶A2（pla2）的活性，当向水溶液中添加甲醇或乙醇时会降低。使用有限差分静电计算来研究模拟混合溶剂对组氨酸48和氨基末端基团的pKa值的影响，这两个基团都会影响催化作用的pH依赖性。计算和实验表明，这些pKa值无法解释活性降低的原因。相对于100%的水性溶剂，在20%的醇类溶剂中，过渡态中的电荷分离不稳定。计算值是稳定和不稳定贡献的组合，与实验结果在定性上一致。饱和介电理论用于模拟高电场中溶剂水的有序排列，并假设在20%醇类水平下，水的介电结构起主导作用。观察到的一致性证明了过渡态稳定理论和连续介质溶剂建模的实用性。进一步表明，对kcat的静电效应有助于解释pH值在7左右时活性的pH依赖性，以及先前报道的电荷突变体的活性变化。
