Damborský J, Bohác M, Prokop M, Kutý M, Koca J
Laboratory of Biomolecular Structure and Dynamics, Faculty of Science, Masaryk University, Brno, Czech Republic.
Protein Eng. 1998 Oct;11(10):901-7. doi: 10.1093/protein/11.10.901.
The application of molecular modelling and quantum-chemistry calculations for the 'computational site-directed mutagenesis' of haloalkane dehalogenase is described here. The exhaustive set of single point mutants of haloalkane dehalogenase in position 172 was constructed by homology modelling. The ability of substituting residues to stabilize the halide ion formed during the dehalogenation reaction in the enzyme active site was probed by quantum-chemical calculations. A simplified modelling procedure was adopted to obtain informative results on the potential activity of mutant proteins in a sufficiently short period of time, which, in the future, could be applicable for making bona fide predictions of mutants' activity prior to their preparation in the laboratory. The reaction pathways for the carbon-halide bond cleavage were calculated using microscopic models of wild type and mutant proteins. The theoretical parameters derived from the calculation, i.e. relative energies and selected atomic charges of educt, product and transition state structures, were statistically correlated with experimentally determined activities. The charge difference of educt and product on the halide-stabilizing hydrogen atom of residue 172 was the best parameter to distinguish protein variants with high activity from mutant proteins displaying a low activity. All mutants with significant activity in the experiment were found to have this parameter one order of magnitude higher than mutants with low activity. The results obtained are discussed in the light of the practical application of this methodology for the prediction of potentially active protein variants. Further automation of the modelling procedure is suggested for combinatorial screening of the large number of protein variants. Coupling of the dehalogenation reaction with hydrogenation of the halide ion formed during the reaction in the enzyme active site was proposed as a possible way to improve the catalytic activity of the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10.
本文描述了分子建模和量子化学计算在卤代烷脱卤酶“计算性定点诱变”中的应用。通过同源建模构建了卤代烷脱卤酶172位的所有单点突变体。通过量子化学计算探究了取代残基稳定酶活性位点脱卤反应过程中形成的卤离子的能力。采用了一种简化的建模程序，以便在足够短的时间内获得有关突变蛋白潜在活性的信息性结果，未来这可用于在实验室制备突变体之前对其活性进行真正的预测。使用野生型和突变蛋白的微观模型计算了碳 - 卤键断裂的反应途径。计算得出的理论参数，即反应物、产物和过渡态结构的相对能量和选定的原子电荷，与实验测定的活性进行了统计关联。172位残基的卤离子稳定氢原子上反应物和产物的电荷差是区分高活性蛋白变体与低活性突变蛋白的最佳参数。实验中所有具有显著活性的突变体的该参数都比低活性突变体高一个数量级。根据该方法在预测潜在活性蛋白变体方面的实际应用对所得结果进行了讨论。建议进一步自动化建模程序以对大量蛋白变体进行组合筛选。提出将脱卤反应与酶活性位点反应过程中形成的卤离子的氢化反应耦合，作为提高自养黄色杆菌GJ10卤代烷脱卤酶催化活性的一种可能方法。
