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Identification of the Ionization State and pKa for Protonation of the 4′‐Aminopyrimidine Ring on Enzymes Utilizing Thiamin Diphosphate by Circular Dichroism Spectroscopy
Author(s) -
Nemeria Natalia,
Yep Alejandra,
McLeish Michael,
Kenyon Geoge L,
Jordan Frank
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1016
Subject(s) - chemistry , protonation , tautomer , circular dichroism , stereochemistry , moiety , enzyme , crystallography , organic chemistry , ion
At Rutgers, three forms of the 4′‐aminopyrimidine moiety of enzyme‐bound thiamin diphosphate (ThDP) were proposed. Circular dichroism (CD) signatures were assigned to the 4′‐aminopyrimidine tautomer (AP; negative band at 320–330 nm) and the 1′, 4′ iminopyrimidine tautomer (IP; positive band at 300–314 nm), no signature is known for the 4′‐aminopyrimidinium form (APH + ). Recently (Nemeria et al., PNAS USA in press), the AP and IP forms were identified on some enzymes in the absence of substrate analogues, the IP form could be induced when the first covalent tetrahedral intermediate on ThDP is formed. We report the pH titration of the AP form observed in the CD spectra of both benzaldehyde lyase and benzoylformate decarboxylase, revealing the transition for the APH + = AP equilibrium providing pK a s of 7.4 and 7.5 for the enzymes, respectively. This constitutes the first direct determination of this pK a on any ThDP‐dependent enzyme, indicating that the enzymes raise the pK a from the aqueous value of 4.85, presumably via stabilization of the APH + form by the highly conserved glutamate within hydrogen bonding distance of the N1′ atom of ThDP. Supported by NIH GM‐050380 (NJ) and NSF EF‐0425719 (MI).