Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 19 April 1985:
Vol. 228. no. 4697, pp. 291 - 297
DOI: 10.1126/science.3838593
Prev | Table of Contents | Next

Articles

Science, Vol 228, Issue 4697, 291-297
Copyright © 1985 by American Association for the Advancement of Science

articles

Redesigning trypsin: alteration of substrate specificity

CS Craik, C Largman, T Fletcher, S Roczniak, PJ Barr, R Fletterick, and WJ Rutter

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.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Autosomal Ichthyosis with Hypotrichosis Syndrome Displays Low Matriptase Proteolytic Activity and Is Phenocopied in ST14 Hypomorphic Mice.
K. List, B. Currie, T. C. Scharschmidt, R. Szabo, J. Shireman, A. Molinolo, B. F. Cravatt, J. Segre, and T. H. Bugge (2007)
J. Biol. Chem. 282, 36714-36723
   Abstract »    Full Text »    PDF »
Duplication and Divergence of 2 Distinct Pancreatic Ribonuclease Genes in Leaf-Eating African and Asian Colobine Monkeys.
J. E. Schienman, R. A. Holt, M. R. Auerbach, and C.-B. Stewart (2006)
Mol. Biol. Evol. 23, 1465-1479
   Abstract »    Full Text »    PDF »
Trypsin-2 Degrades Human Type II Collagen and Is Expressed and Activated in Mesenchymally Transformed Rheumatoid Arthritis Synovitis Tissue.
M. Stenman, M. Ainola, L. Valmu, A. Bjartell, G. Ma, U.-H. Stenman, T. Sorsa, R. Luukkainen, and Y. T. Konttinen (2005)
Am. J. Pathol. 167, 1119-1124
   Abstract »    Full Text »    PDF »
The Identification of Residues That Control Signal Peptidase Cleavage Fidelity and Substrate Specificity.
A. Karla, M. O. Lively, M. Paetzel, and R. Dalbey (2005)
J. Biol. Chem. 280, 6731-6741
   Abstract »    Full Text »    PDF »
The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274-His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion.
L. Zhao, H. Liao, and M.-D. Tsai (2004)
J. Biol. Chem. 279, 31995-32000
   Abstract »    Full Text »    PDF »
Ala226 to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease.
B. Jelinek, J. Antal, I. Venekei, and L. Graf (2004)
Protein Eng. Des. Sel. 17, 127-131
   Abstract »    Full Text »    PDF »
Evolution of Trypsinogen Activation Peptides.
J.-M. Chen, Z. Kukor, C. Le Marechal, M. Toth, L. Tsakiris, O. Raguenes, C. Ferec, and M. Sahin-Toth (2003)
Mol. Biol. Evol. 20, 1767-1777
   Abstract »    Full Text »    PDF »
Engineering the proteolytic specificity of activated protein C improves its pharmacological properties.
D. T. Berg, B. Gerlitz, J. Shang, T. Smith, P. Santa, M. A. Richardson, K. D. Kurz, B. W. Grinnell, K. Mace, and B. E. Jones (2003)
PNAS 100, 4423-4428
   Abstract »    Full Text »    PDF »
Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype.
S. Akhavan, R. De Cristofaro, F. Peyvandi, S. Lavoretano, R. Landolfi, and P. M. Mannucci (2002)
Blood 100, 1347-1353
   Abstract »    Full Text »    PDF »
Hydrolysis by Cathepsin B of Fluorescent Peptides Derived From Human Prorenin.
P. C. Almeida, V. Oliveira, J. R. Chagas, M. Meldal, M. A. Juliano, and L. Juliano (2000)
Hypertension 35, 1278-1283
   Abstract »    Full Text »    PDF »
Mutational Analysis of the Primary Substrate Specificity Pocket of Complement Factor B. ASP226 IS A MAJOR STRUCTURAL DETERMINANT FOR P1-ARG BINDING.
Y. Xu, A. Circolo, H. Jing, Y. Wang, S. V. L. Narayana, and J. E. Volanakis (2000)
J. Biol. Chem. 275, 378-385
   Abstract »    Full Text »    PDF »
Unexpected crucial role of residue 225 in serine proteases.
E. R. Guinto, S. Caccia, T. Rose, K. Futterer, G. Waksman, and E. Di Cera (1999)
PNAS 96, 1852-1857
   Abstract »    Full Text »    PDF »
A streptavidin mutant with altered ligand-binding specificity.
G. O. Reznik, S. Vajda, T. Sano, and C. R. Cantor (1998)
PNAS 95, 13525-13530
   Abstract »    Full Text »    PDF »
Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR.
D. P. Chandler, C. A. Wagnon, and H. Bolton Jr. (1998)
Appl. Envir. Microbiol. 64, 669-677
   Abstract »    Full Text »
Evolutionary Divergence of Substrate Specificity within the Chymotrypsin-like Serine Protease Fold.
J. J. Perona and C. S. Craik (1997)
J. Biol. Chem. 272, 29987-29990
   Full Text »    PDF »
Substrate Recognition by Recombinant Serine Collagenase 1 from Uca pugilator.
C. A. Tsu and C. S. Craik (1996)
J. Biol. Chem. 271, 11563-11570
   Abstract »    Full Text »    PDF »
Crystal structure of a catalytic antibody with a serine protease active site.
G. Zhou, J Guo, W Huang, R. Fletterick, and T. Scanlan (1994)
Science 265, 1059-1064
   Abstract »    PDF »
Control of enzyme activity by an engineered disulfide bond.
M Matsumura and B. Matthews (1989)
Science 243, 792-794
   Abstract »    PDF »
Computer-aided molecular design.
J. McCammon (1987)
Science 238, 486-491
   Abstract »    PDF »
The catalytic role of the active site aspartic acid in serine proteases.
C. Craik, S Roczniak, C Largman, and W. Rutter (1987)
Science 237, 909-913
   Abstract »    PDF »
Engineering enzyme specificity by "substrate-assisted catalysis".
P Carter and J. Wells (1987)
Science 237, 394-399
   Abstract »    PDF »
Tinkering with enzymes: what are we learning?.
J. Knowles (1987)
Science 236, 1252-1258
   Abstract »    PDF »
Evolution of Catalysis in the Serine Proteases.
J.N. Higaki, B.W. Gibson, and C.S. Craik (1987)
Cold Spring Harb Symp Quant Biol 52, 615-621
   Abstract »    PDF »
Sequence Convergence and Functional Adaptation of Stomach Lysozymes from Foregut Fermenters.
C.-B. Stewart and A.C. Wilson (1987)
Cold Spring Harb Symp Quant Biol 52, 891-899
   Abstract »    PDF »
Probing Steric and Hydrophobic Effects on Enzyme-Substrate Interactions by Protein Engineering.
D. A. ESTELL, T. P. GRAYCAR, J. V. MILLER, D. B. POWERS, J. A. WELLS, J. P. BURNIER, and P. G. NG (1986)
Science 233, 659-663
   Abstract »    PDF »
Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis.
E. Howell, J. Villafranca, M. Warren, S. Oatley, and J Kraut (1986)
Science 231, 1123-1128
   Abstract »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)