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.

[1]  D. Agard,et al.  Structural analysis of specificity: .alpha.-lytic protease complexes with analogs of reaction intermediates , 1989 .

[2]  G. Náray‐Szabó.  Quantitative estimation of activities of mutant enzymes , 1989 .

[3]  U. Singh,et al.  Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin , 1987, Nature.

[4]  A. Fersht,et al.  Hydrogen bonding and biological specificity analysed by protein engineering , 1985, Nature.

[5]  K. Martínek,et al.  The specificity in the elementary steps of alpha-chymotrypsin catalysis. A temperature study with a series of N-acetyl-L-amino acid methyl esters. , 1982, Biochimica et biophysica acta.

[6]  A. Warshel,et al.  Evaluation of catalytic free energies in genetically modified proteins. , 1988, Journal of molecular biology.

[7]  A. D. McLachlan,et al.  Solvation energy in protein folding and binding , 1986, Nature.

[8]  D. Agard,et al.  Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids. , 1988, Biochemistry.

[9]  P. Kollman,et al.  Free energy calculations on binding and catalysis by α‐lytic protease: The role of substrate size in the P1 pocket , 1991, Proteins: Structure, Function, and Bioinformatics.

[10]  A. Shrake,et al.  Environment and exposure to solvent of protein atoms. Lysozyme and insulin. , 1973, Journal of molecular biology.

[11]  J. Ponder,et al.  Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. , 1987, Journal of molecular biology.

[12]  L. Delbaere,et al.  Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences. , 1978, Canadian journal of biochemistry.

[13]  D. Agard,et al.  Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters. , 1993, Biochemistry.

[14]  R. Bruccoleri,et al.  On the attribution of binding energy in antigen-antibody complexes McPC 603, D1.3, and HyHEL-5. , 1989, Biochemistry.

[15]  X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state. , 1975, The Journal of biological chemistry.

[16]  D. Agard,et al.  Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates. , 1990, Biochemistry.

[17]  D. Agard,et al.  Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid. , 1987, Biochemistry.

[18]  David A. Agard,et al.  Structural plasticity broadens the specificity of an engineered protease , 1989, Nature.

[19]  D A Agard,et al.  Structural basis for broad specificity in alpha-lytic protease mutants. , 1993, Biochemistry.

[20]  U. Singh,et al.  A NEW FORCE FIELD FOR MOLECULAR MECHANICAL SIMULATION OF NUCLEIC ACIDS AND PROTEINS , 1984 .

[21]  P. A. Bash,et al.  Free energy calculations by computer simulation. , 1987, Science.

[22]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.