The Role of Loop-Loop Interactions in Coordinating Motions and Enzymatic Function in Triosephosphate Isomerase
Identifieur interne : 003791 ( Main/Exploration ); précédent : 003790; suivant : 003792The Role of Loop-Loop Interactions in Coordinating Motions and Enzymatic Function in Triosephosphate Isomerase
Auteurs : Yan Wang [États-Unis] ; Rebecca B. Berlow [États-Unis] ; J. Patrick Loria [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2009.
Abstract
The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homolog caused a 102-fold loss in enzymatic activity, a decrease in substrate binding affinity and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional 1H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R1ρ experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and the coordinated motion of loop 6 is reduced relative to WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur, and that the interactions play a significant role in modulating the chemical dynamics near the active site.
Url:
DOI: 10.1021/bi9002887
PubMed: 19348462
PubMed Central: 2713366
Affiliations:
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Berlow</name><affiliation wicri:level="2"><nlm:aff id="A2">Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea>Department of Molecular Biophysics and Biochemistry, Yale University, New Haven</wicri:cityArea></affiliation></author><author><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><affiliation wicri:level="2"><nlm:aff id="A1">Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea>Department of Chemistry, Yale University, New Haven</wicri:cityArea></affiliation><affiliation wicri:level="2"><nlm:aff id="A2">Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea>Department of Molecular Biophysics and Biochemistry, Yale University, New Haven</wicri:cityArea></affiliation></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homolog caused a 10<sup>2</sup>-fold loss in enzymatic activity, a decrease in substrate binding affinity and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional <sup>1</sup>H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R<sub>1ρ</sub> experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and the coordinated motion of loop 6 is reduced relative to WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur, and that the interactions play a significant role in modulating the chemical dynamics near the active site.</p></div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Connecticut</li></region></list><tree><country name="États-Unis"><region name="Connecticut"><name sortKey="Wang, Yan" sort="Wang, Yan" uniqKey="Wang Y" first="Yan" last="Wang">Yan Wang</name></region><name sortKey="Berlow, Rebecca B" sort="Berlow, Rebecca B" uniqKey="Berlow R" first="Rebecca B." last="Berlow">Rebecca B. Berlow</name><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. 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