Correlation of the enzyme activities of Bacillus stearothermophilus lactate dehydrogenase on three substrates with the results of molecular dynamics energy minimization conformational searching

T R Dafforn, I G Badcoe, R B Sessions, A S El-Hawrani, J J Holbrook

Research output: Contribution to journalArticle (Academic Journal)peer-review


Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15-Angstrom sphere of the ternary complex centered on the catalytic histidine, The search is carried out by calculating a 30-ps dynamics trajectory at 300 K and minimizing structures at 1-ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant /wt LDH, In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate-active site interactions were measured in each of the 420 minimized structures, These parameters were then compared to the measured catalytic activity of the protein-substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodolgy it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis. (C) 1997 Wiley-Liss, Inc.

Original languageEnglish
Pages (from-to)228-239
Number of pages12
JournalProteins: Structure, Function, and Bioinformatics
Issue number2
Publication statusPublished - Oct 1997

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