Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase.
GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed. Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response. We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail. The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors. Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2. The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior. The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme complex.
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Laura E
LE
Naught
Sunny
S
Gilbert
Rebecca
R
Imhoff
Christopher
C
Snook
Lesa
L
Beamer
Peter
P
Tipton
D000494:Allosteric Regulation; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation
D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase