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Article: Direct measurement of the pK(a) of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: Implications for the catalytic mechanism

TitleDirect measurement of the pK(a) of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: Implications for the catalytic mechanism
Authors
Issue Date1999
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry
Citation
Biochemistry, 1999, v. 38 n. 25, p. 8038-8044 How to Cite?
AbstractThe ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cγ] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pK(a) greater than ~4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pK(a) value of less than 4 in this complex. Such a low pK(a) value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pK(a)=6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 13Cγ resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.
Persistent Identifierhttp://hdl.handle.net/10722/157305
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorCasarotto, MGen_US
dc.contributor.authorBasran, Jen_US
dc.contributor.authorBadii, Ren_US
dc.contributor.authorSze, KHen_US
dc.contributor.authorRoberts, GCKen_US
dc.date.accessioned2012-08-08T08:48:48Z-
dc.date.available2012-08-08T08:48:48Z-
dc.date.issued1999en_US
dc.identifier.citationBiochemistry, 1999, v. 38 n. 25, p. 8038-8044en_US
dc.identifier.issn0006-2960en_US
dc.identifier.urihttp://hdl.handle.net/10722/157305-
dc.description.abstractThe ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cγ] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pK(a) greater than ~4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pK(a) value of less than 4 in this complex. Such a low pK(a) value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pK(a)=6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 13Cγ resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.en_US
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_US
dc.relation.ispartofBiochemistryen_US
dc.subject.meshApoenzymes - Chemistry - Metabolismen_US
dc.subject.meshAspartic Acid - Chemistry - Metabolismen_US
dc.subject.meshCatalysisen_US
dc.subject.meshFolic Acid - Analogs & Derivatives - Chemistry - Metabolismen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshLactobacillus Casei - Enzymologyen_US
dc.subject.meshMacromolecular Substancesen_US
dc.subject.meshNad - Analogs & Derivatives - Chemistry - Metabolismen_US
dc.subject.meshTetrahydrofolate Dehydrogenase - Chemistry - Metabolismen_US
dc.titleDirect measurement of the pK(a) of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: Implications for the catalytic mechanismen_US
dc.typeArticleen_US
dc.identifier.emailSze, KH:khsze@hku.hken_US
dc.identifier.authoritySze, KH=rp00785en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1021/bi990301pen_US
dc.identifier.pmid10387048-
dc.identifier.scopuseid_2-s2.0-0033594816en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033594816&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume38en_US
dc.identifier.issue25en_US
dc.identifier.spage8038en_US
dc.identifier.epage8044en_US
dc.identifier.isiWOS:000081138400019-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridCasarotto, MG=6701682753en_US
dc.identifier.scopusauthoridBasran, J=7003996757en_US
dc.identifier.scopusauthoridBadii, R=6603978077en_US
dc.identifier.scopusauthoridSze, KH=7006735061en_US
dc.identifier.scopusauthoridRoberts, GCK=7403400348en_US
dc.identifier.citeulike4472287-

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