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Correlation of the enzyme activities of Bacillus stearothermophilus lactate dehydrogenase on three substrates with the results of molecular dynamics/energy minimization conformational searching
Author(s) -
Dafforn Timothy R.,
Badcoe Ian G.,
Sessions Richard B.,
El Hawrani Ayman S.,
Holbrook J. John
Publication year - 1997
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(199710)29:2<228::aid-prot10>3.0.co;2-q
Subject(s) - molecular dynamics , substrate (aquarium) , ternary complex , energy minimization , lactate dehydrogenase , histidine , chemistry , active site , protein engineering , biological system , enzyme , stereochemistry , crystallography , biophysics , computational chemistry , biochemistry , biology , ecology
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 1.1.1.27) 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‐Å 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. Proteins 29:228–239, 1997. © 1997 Wiley‐Liss, Inc.