Abstract
A preliminary attempt to simulate the observed effect of a site-directed mutagenesis of rat trypsin gives encouraging results. The calculations reproduce in a semiquantitative way the observed change in the activation barrier of the rate-limiting step of amide hydrolysis. This result, which did not require any adjustable parameters, indicates that our method may provide a reliable basis for computer-aided enzyme design. In addition to the potentially practical value of the calculations, they provide important mechanistic information--that is, the change in the catalytic effect in trypsin appears to be almost exclusively due to the change in the electrostatic stabilization of the ionic configurations. This supports the view that electrostatic effects are the major factor in enzyme catalysis.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blow D. M., Birktoft J. J., Hartley B. S. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature. 1969 Jan 25;221(5178):337–340. doi: 10.1038/221337a0. [DOI] [PubMed] [Google Scholar]
- Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
- Dalbadie-McFarland G., Cohen L. W., Riggs A. D., Morin C., Itakura K., Richards J. H. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409–6413. doi: 10.1073/pnas.79.21.6409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huber R., Kukla D., Bode W., Schwager P., Bartels K., Deisenhofer J., Steigemann W. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. J Mol Biol. 1974 Oct 15;89(1):73–101. doi: 10.1016/0022-2836(74)90163-6. [DOI] [PubMed] [Google Scholar]
- Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
- Kraut J. Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem. 1977;46:331–358. doi: 10.1146/annurev.bi.46.070177.001555. [DOI] [PubMed] [Google Scholar]
- Levitt M. Protein conformation, dynamics, and folding by computer simulation. Annu Rev Biophys Bioeng. 1982;11:251–271. doi: 10.1146/annurev.bb.11.060182.001343. [DOI] [PubMed] [Google Scholar]
- Russell S. T., Warshel A. Calculations of electrostatic energies in proteins. The energetics of ionized groups in bovine pancreatic trypsin inhibitor. J Mol Biol. 1985 Sep 20;185(2):389–404. doi: 10.1016/0022-2836(85)90411-5. [DOI] [PubMed] [Google Scholar]
- Villafranca J. E., Howell E. E., Voet D. H., Strobel M. S., Ogden R. C., Abelson J. N., Kraut J. Directed mutagenesis of dihydrofolate reductase. Science. 1983 Nov 18;222(4625):782–788. doi: 10.1126/science.6356360. [DOI] [PubMed] [Google Scholar]
- Wang D., Bode W., Huber R. Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution. J Mol Biol. 1985 Oct 5;185(3):595–624. doi: 10.1016/0022-2836(85)90074-9. [DOI] [PubMed] [Google Scholar]
- Warshel A. Dynamics of enzymatic reactions. Proc Natl Acad Sci U S A. 1984 Jan;81(2):444–448. doi: 10.1073/pnas.81.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Warshel A., Russell S. T. Calculations of electrostatic interactions in biological systems and in solutions. Q Rev Biophys. 1984 Aug;17(3):283–422. doi: 10.1017/s0033583500005333. [DOI] [PubMed] [Google Scholar]
- Wilkinson A. J., Fersht A. R., Blow D. M., Carter P., Winter G. A large increase in enzyme-substrate affinity by protein engineering. Nature. 1984 Jan 12;307(5947):187–188. doi: 10.1038/307187a0. [DOI] [PubMed] [Google Scholar]