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.

Site Tools

  • AAAS
  • Subscribe
  • Feedback

Site Search

Search Advanced

Science 8 August 1986:
Vol. 233. no. 4764, pp. 659 - 663
DOI: 10.1126/science.233.4764.659
Prev | Table of Contents | Next

Articles

Probing Steric and Hydrophobic Effects on Enzyme-Substrate Interactions by Protein Engineering

D. A. ESTELL 1, T. P. GRAYCAR 1, J. V. MILLER 1, D. B. POWERS 2, J. A. WELLS 2, J. P. BURNIER 3, and P. G. NG 3

1 Research Department, Genencor, Inc., 180 Kimball Way, South San Francisco, CA 94080.
2 Department of Biocatalysis, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080.
3 Department of Developmental Biology, Genentech, South San Francisco, CA 94080.

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly166), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (kcat/Km) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft (sim160 Å3), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (kcat/Km) (up to 5000 times) as a result of steric hindrance.

Submitted on February 18, 1986
Accepted on June 5, 1986


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Roles of S3 Site Residues of Nattokinase on its Activity and Substrate Specificity.
S. Wu, C. Feng, J. Zhong, and L. Huan (2007)
J. Biochem. 142, 357-364
   Abstract »    Full Text »    PDF »
Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'.
P. J. Ganz, M. D. Bauer, Y. Sun, A. M. Fieno, R. A. Grant, P. E. Correa, M. Laskowski Jr, and C. W. Saunders (2004)
Protein Eng. Des. Sel. 17, 333-339
   Abstract »    Full Text »    PDF »
Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence.
Y. Kannan, Y. Koga, Y. Inoue, M. Haruki, M. Takagi, T. Imanaka, M. Morikawa, and S. Kanaya (2001)
Appl. Envir. Microbiol. 67, 2445-2452
   Abstract »    Full Text »
Molecular and Biotechnological Aspects of Microbial Proteases.
M. B. Rao, A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande (1998)
Microbiol. Mol. Biol. Rev. 62, 597-635
   Abstract »    Full Text »    PDF »
Assembly of an active enzyme by the linkage of two protein modules.
A. E. Nixon, M. S. Warren, and S. J. Benkovic (1997)
PNAS 94, 1069-1073
   Abstract »    Full Text »    PDF »
Mutational Anatomy of an HIV-1 Protease Variant Conferring Cross-resistance to Protease Inhibitors in Clinical Trials. COMPENSATORY MODULATIONS OF BINDING AND ACTIVITY.
H. B. Schock, V. M. Garsky, and L. C. Kuo (1996)
J. Biol. Chem. 271, 31957-31963
   Abstract »    Full Text »    PDF »
Substrate phage: selection of protease substrates by monovalent phage display.
D. Matthews and J. Wells (1993)
Science 260, 1113-1117
   Abstract »    PDF »
A general method for site-specific incorporation of unnatural amino acids into proteins.
C. Noren, S. Anthony-Cahill, M. Griffith, and P. Schultz (1989)
Science 244, 182-188
   Abstract »    PDF »
Direct Bronsted analysis of the restoration of activity to a mutant enzyme by exogenous amines.
M. Toney and J. Kirsch (1989)
Science 243, 1485-1488
   Abstract »    PDF »
The interplay between chemistry and biology in the design of enzymatic catalysts.
P. Schultz (1988)
Science 240, 426-433
   Abstract »    PDF »
Insights into enzyme function from studies on mutants of dihydrofolate reductase.
S. Benkovic, C. Fierke, and A. Naylor (1988)
Science 239, 1105-1110
   Abstract »    PDF »
Generation of a hybrid sequence-specific single-stranded deoxyribonuclease.
D. Corey and P. Schultz (1987)
Science 238, 1401-1403
   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 »
On the Evolution of Specificity and Catalysis in Subtilisin.
J.A. Wells, B.C. Cunningham, T.P. Graycar, D.A. Estell, and P. Carter (1987)
Cold Spring Harb Symp Quant Biol 52, 647-652
   Abstract »    PDF »


To Advertise     Find Products

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