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 24 July 1987:
Vol. 237. no. 4813, pp. 394 - 399
DOI: 10.1126/science.3299704
Prev | Table of Contents | Next

Articles

Science, Vol 237, Issue 4813, 394-399
Copyright © 1987 by American Association for the Advancement of Science

articles

Engineering enzyme specificity by "substrate-assisted catalysis"

P Carter and JA Wells

A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His with Ala in the Bacillus amyloliquefaciens subtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed with N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) at pH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependent pH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
In Vivo Reshaping the Catalytic Site of Nucleoside 2'-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution.
P. A. Kaminski, P. Dacher, L. Dugue, and S. Pochet (2008)
J. Biol. Chem. 283, 20053-20059
   Abstract »    Full Text »    PDF »
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 »
Transcript-assisted transcriptional proofreading..
N. Zenkin, Y. Yuzenkova, and K. Severinov (2006)
Science 313, 518-520
   Abstract »    Full Text »    PDF »
PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase.
S. Blanga-Kanfi, M. Amitsur, A. Azem, and G. Kaufmann (2006)
Nucleic Acids Res. 34, 3209-3219
   Abstract »    Full Text »    PDF »
The Inhibitory {gamma} Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure.
L.-W. Guo, H. Muradov, A. R. Hajipour, M. K. Sievert, N. O. Artemyev, and A. E. Ruoho (2006)
J. Biol. Chem. 281, 15412-15422
   Abstract »    Full Text »    PDF »
Chemical rescue of a mutant enzyme in living cells..
Y. Qiao, H. Molina, A. Pandey, J. Zhang, and P. A. Cole (2006)
Science 311, 1293-1297
   Abstract »    Full Text »    PDF »
Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a.
S. A. McCartney, E. J. Brignole, K. N. Kolegraff, A. N. Loveland, L. M. Ussin, and W. Gibson (2005)
J. Biol. Chem. 280, 33206-33212
   Abstract »    Full Text »    PDF »
Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity.
N. Varadarajan, J. Gam, M. J. Olsen, G. Georgiou, and B. L. Iverson (2005)
PNAS 102, 6855-6860
   Abstract »    Full Text »    PDF »
Evidence for Distinct Roles in Catalysis for Residues of the Serine-Serine-Lysine Catalytic Triad of Fatty Acid Amide Hydrolase.
M. K. McKinney and B. F. Cravatt (2003)
J. Biol. Chem. 278, 37393-37399
   Abstract »    Full Text »    PDF »
Zeptomole detection of a viral nucleic acid using a target-activated ribozyme.
N. K. VAISH, V. R. JADHAV, K. KOSSEN, C. PASKO, L. E. ANDREWS, J. A. MCSWIGGEN, B. POLISKY, and S. D. SEIWERT (2003)
RNA 9, 1058-1072
   Abstract »    Full Text »    PDF »
From the Cover: A clogged gutter mechanism for protease inhibitors.
E. S. Radisky and D. E. Koshland Jr. (2002)
PNAS 99, 10316-10321
   Abstract »    Full Text »    PDF »
Selection of catalytically active biotin ligase and trypsin mutants by phage display.
C. Heinis, A. Huber, S. Demartis, J. Bertschinger, S. Melkko, L. Lozzi, P. Neri, and D. Neri (2001)
Protein Eng. Des. Sel. 14, 1043-1052
   Abstract »    Full Text »    PDF »
Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression.
G. E. Sroga and J. S. Dordick (2001)
Protein Eng. Des. Sel. 14, 929-937
   Abstract »    Full Text »    PDF »
Elastase substrate specificity tailored through substrate-assisted catalysis and phage display.
W. Dall'Acqua, C. Halin, M. L. Rodrigues, and P. Carter (1999)
Protein Eng. Des. Sel. 12, 981-987
   Abstract »    Full Text »    PDF »
Efficient site specific removal of a C-terminal FLAG fusion from a Fab' using copper(II) ion catalysed protein cleavage.
D. P. Humphreys, B. J. Smith, L. M. King, S. M. West, D. G. Reeks, and P. E. Stephens (1999)
Protein Eng. Des. Sel. 12, 179-184
   Abstract »    Full Text »    PDF »
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 »
Substrate phage: selection of protease substrates by monovalent phage display.
D. Matthews and J. Wells (1993)
Science 260, 1113-1117
   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 »
Control of enzyme activity by an engineered disulfide bond.
M Matsumura and B. Matthews (1989)
Science 243, 792-794
   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 »
Chemical Rescue of a Mutant Protein-tyrosine Kinase.
D. M. Williams, D. Wang, and P. A. Cole (2000)
J. Biol. Chem. 275, 38127-38130
   Abstract »    Full Text »    PDF »
Clarifying the Catalytic Roles of Conserved Residues in the Amidase Signature Family.
M. P. Patricelli and B. F. Cravatt (2000)
J. Biol. Chem. 275, 19177-19184
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

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