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Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases

Gershenson, Anne and Schauerte, Joseph A. and Giver, Lori and Arnold, Frances H. (2000) Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases. Biochemistry, 39 (16). pp. 4658-4665. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170201-092852593

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Abstract

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 °C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi992473sDOIArticle
http://pubs.acs.org/doi/full/10.1021/bi992473sPublisherArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2000 American Chemical Society. Received 25 October 1999. Published online 30 March 2000. Published in print 1 April 2000. This work was supported by ARO Grant DAAH04-95-1-0613 (F.H.A.) and by National Institute of Aging Grant AG09761 (J.A.S.). We thank Dr. Patrick L. Wintrode for helpful discussions and comments. We also thank Dr. Kentaro Miyazki and Christopher J. Fischer for access to data prior to publication.
Funders:
Funding AgencyGrant Number
Army Research Office (ARO)DAAH04-95-1-0613
NIHAG09761
National Institute on AgingUNSPECIFIED
Issue or Number:16
Record Number:CaltechAUTHORS:20170201-092852593
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170201-092852593
Official Citation:Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases Anne Gershenson, Joseph A. Schauerte, Lori Giver, and Frances H. Arnold Biochemistry 2000 39 (16), 4658-4665 DOI: 10.1021/bi992473s
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73917
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 18:02
Last Modified:03 Oct 2019 16:32

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