Publication Date:
2009-10-30
Description:
Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K(D)) and the Michaelis constant under single-turnover conditions (K(M)*), and those in the (+) half-site primarily decrease the turnover number (k(cat)*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating K(M)*, whereas redesigns with altered specificities on the (+) side modulate k(cat)*. Our results illustrate how classical enzymology and modern protein design can each inform the other.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771326/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771326/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Thyme, Summer B -- Jarjour, Jordan -- Takeuchi, Ryo -- Havranek, James J -- Ashworth, Justin -- Scharenberg, Andrew M -- Stoddard, Barry L -- Baker, David -- GM084433/GM/NIGMS NIH HHS/ -- R00 RR024107/RR/NCRR NIH HHS/ -- R00 RR024107-03/RR/NCRR NIH HHS/ -- R00 RR024107-04/RR/NCRR NIH HHS/ -- RL1 GM084433/GM/NIGMS NIH HHS/ -- RL1 GM084433-03/GM/NIGMS NIH HHS/ -- RL1CA133832/CA/NCI NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Oct 29;461(7268):1300-4. doi: 10.1038/nature08508.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA. sthyme@u.washington.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19865174" target="_blank"〉PubMed〈/a〉
Keywords:
Binding Sites
;
*Biocatalysis
;
Computational Biology
;
*Computer Simulation
;
DNA/chemistry/metabolism
;
Endonucleases/chemistry/*metabolism
;
Kinetics
;
Models, Molecular
;
Protein Binding
;
Protein Conformation
;
RNA-Directed DNA Polymerase/chemistry/*metabolism
;
Saccharomyces cerevisiae/metabolism
;
Substrate Specificity
;
*Thermodynamics
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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