CORRELATION OF THE ENZYME-ACTIVITIES OF BACILLUS-STEAROTHERMOPHILUS LACTATE-DEHYDROGENASE ON 3 SUBSTRATES WITH THE RESULTS OF MOLECULAR-DYNAMICS ENERGY MINIMIZATION CONFORMATIONAL SEARCHING

Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this ar ticle we detail the use of a molecular mechanics approach for suggesti ng regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of intere st for protein engineers. The approach combines molecular dynamics wit h energy minimization (MD/EM) to search the conformational space avail able to a 15-Angstrom sphere of the ternary complex centered on the ca talytic histidine, The search is carried out by calculating a 30-ps dy namics trajectory at 300 K and minimizing structures at 1-ps intervals . The protocol has been performed on 14 systems containing different c ombinations of substrate and mutant /wt LDH, In order to discover whic h interactions are important in defining substrate specificity, eight conformational parameters representing substrate-active site interacti ons were measured in each of the 420 minimized structures, These param eters were then compared to the measured catalytic activity of the pro tein-substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using th is methodolgy it is possible to estimate the catalytic activity of pro teins of varied sequence by computer simulation before synthesis. (C) 1997 Wiley-Liss, Inc.