Computational method for the design of enzymes with altered substrate specificity.
A combination of enzyme kinetics and X-ray crystallographic analysis of site-specific mutants has been used to probe the determinants of substrate specificity for the enzyme alpha-lytic protease. We now present a generalized model for understanding the effects of mutagenesis on enzyme substrate specificity. This algorithm uses a library of side-chain rotamers to sample conformation space within the binding site for the enzyme-substrate complex. The free energy of each conformation is evaluated with a standard molecular mechanics force field, modified to include a solvation energy term. This rapid energy calculation based on coarse conformation sampling quite accurately predicts the relative catalytic efficiency of over 40 different alpha-lytic protease-substrate combinations. Unlike other computational approaches, with this method it is feasible to evaluate all possible mutations within the binding site. Using this algorithm, we have successfully designed a protease that is both highly active and selective for a non-natural substrate. These encouraging results indicate that it is possible to design altered enzymes solely on the basis of empirical energy calculations.
Wilson
C
C
Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Mace
J E
JE
Agard
D A
DA
D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014961:X-Ray Diffraction
D004798:Enzymes; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase