Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate.
UDP-galactopyranose mutase is a flavoprotein which catalyses the interconversion of UDP-galactopyranose and UDP-galactofuranose. The enzyme is of interest because it provides the activated biosynthetic precursor of galactofuranose, a key cell wall component of many bacterial pathogens. The reaction mechanism of this mutase is intriguing because the anomeric oxygen forms a glycosidic bond, which means that the reaction must proceed by a novel mechanism involving ring breakage and closure. The structure of the enzyme is known, but the mechanism, although speculated on, is not resolved. The overall reaction is electrically neutral but a crypto-redox reaction is suggested by the requirement that the flavin must adopt the reduced form for activity. Herein we report a thermodynamic analysis of the enzyme's flavin cofactor with the objective of defining the system and setting parameters for possible reaction schemes. The analysis shows that the neutral semiquinone (FADH(*)) is stabilized in the presence of substrate and the fully reduced flavin is the anionic FADH(-) rather than the neutral FADH(2). The anionic FADH(-) has the potential to act as a rapid 1-electron donor/acceptor without being slowed by a coupled proton transfer and is therefore an ideal crypto-redox cofactor.
Fullerton
Stephen W B
SW
Centre for Biomolecular Sciences, The University, St. Andrews KY16 9ST, UK.
Daff
Simon
S
Sanders
David A R
DA
Ingledew
W John
WJ
Whitfield
Chris
C
Chapman
Stephen K
SK
Naismith
James H
JH
D001426:Bacterial Proteins / Q000737:chemistry; D016227:Benzoquinones / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005609:Free Radicals / Q000737:chemistry; D019751:Intramolecular Transferases / Q000737:chemistry*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008024:Ligands; D010084:Oxidation-Reduction; D011199:Potentiometry / Q000379:methods; D011809:Quinones / Q000737:chemistry*; D013379:Substrate Specificity; D013816:Thermodynamics; D014531:Uridine Diphosphate Galactose / Q000737:chemistry
D001426:Bacterial Proteins; D016227:Benzoquinones; D005609:Free Radicals; D008024:Ligands; D011809:Quinones; C001636:flavosemiquinone; D014531:Uridine Diphosphate Galactose; C025232:semiquinone radicals; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase