Related ArticlesThe pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase.
Biochemistry. 1996 Aug 6;35(31):9958-66
Authors: McIntosh LP, Hand G, Johnson PE, Joshi MD, Körner M, Plesniak LA, Ziser L, Wakarchuk WW, Withers SG
The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.
[NMR paper] Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, an
Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.
Related Articles Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.
Biochemistry. 2005 May 24;44(20):7414-26
Authors: Chen TS, Chung FY, Tjong SC, Goh KS, Huang WN, Chien KY, Wu PL, Lin HC, Chen CJ, Wu WG
Natural homologues of cobra cardiotoxins (CTXs) were...
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[NMR paper] Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin
Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR.
Related Articles Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR.
Protein Sci. 2003 Apr;12(4):794-810
Authors: Day RM, Thalhauser CJ, Sudmeier JL, Vincent MP, Torchilin EV, Sanford DG, Bachovchin CW, Bachovchin WW
We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a...
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[NMR paper] Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR.
Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR.
J Mol Biol. 1997 Sep 5;271(5):803-18
Authors: Nieba-Axmann SE, Ottiger M, Wüthrich K, Plückthun A
GroE, the chaperonin system of Escherichia coli, prevents the aggregation of partially folded or misfolded proteins by complexing them in a form competent for...
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[NMR paper] Individual ionization constants of all the carboxyl groups in ribonuclease HI from Es
Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.
Related Articles Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.
Biochemistry. 1994 May 3;33(17):5275-84
Authors: Oda Y, Yamazaki T, Nagayama K, Kanaya S, Kuroda Y, Nakamura H
All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme...
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[NMR paper] Individual ionization constants of all the carboxyl groups in ribonuclease HI from Es
Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.
Related Articles Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.
Biochemistry. 1994 May 3;33(17):5275-84
Authors: Oda Y, Yamazaki T, Nagayama K, Kanaya S, Kuroda Y, Nakamura H
All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme...
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[NMR paper] Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximatel
Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximately 48-kDa insulin receptor protein-tyrosine kinase results in slower rates of diphosphorylation of a series of dodecapeptide substrates. An assessment by 1H NMR.
Related Articles Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximately 48-kDa insulin receptor protein-tyrosine kinase results in slower rates of diphosphorylation of a series of dodecapeptide substrates. An assessment by 1H NMR.
J Biol Chem. 1991 Jul 5;266(19):12369-71
Authors: ...
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[NMR paper] NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylam
NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylamide-DOTA.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylamide-DOTA.
Bioconjug Chem. 1999 May-Jun;10(3):454-63
Authors: Keire DA, Kobayashi M
The conjugation of a chelating agent to a protein via a covalent linkage has been previously reported to change the metal-binding characteristics of the chelator. A fundamental...
The pKa of the general acid/base carboxyl group of a glycosidase cycles during cataly
Linear Mode
