title: Correlation of the enzyme activities of Bacillus stearothermophilus lactate dehydrogenase on three substrates with the results of molecular dynamics/energy minimization conformational searching.
abstract: 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-A 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 methodology it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis.
authors.affiliation: Molecular Recognition Centre, Medical School, University of Bristol, U.K.
authors.forename: T R
authors.initials: TR
authors.lastname: Dafforn
authors.forename: I G
authors.initials: IG
authors.lastname: Badcoe
authors.forename: R B
authors.initials: RB
authors.lastname: Sessions
authors.forename: A S
authors.initials: AS
authors.lastname: el Hawrani
authors.forename: J J
authors.initials: JJ
authors.lastname: Holbrook
mesh_terms: D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D007770:L-Lactate Dehydrogenase