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Redesigning Trypsin: Alteration of Substrate Specificity
Charles S. Craik, Corey Largman, Thomas Fletcher, Steven Roczniak, Philip J. Barr, Robert Fletterick and William J. Rutter
New Series, Vol. 228, No. 4697 (Apr. 19, 1985), pp. 291-297
Published by: American Association for the Advancement of Science
Stable URL: http://www.jstor.org/stable/1694832
Page Count: 7
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A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.
Science © 1985 American Association for the Advancement of Science