Lysine 183 Is the General Base in the 6-Phosphogluconate Dehydrogenase-Catalyzed Reaction

Site-directed mutagenesis was used to change K183 of sheep liver 6-phosphogluconate dehydrogenase to A, E, H, C, Q, R, and M to probe its possible role as a general base catalyst. Each of the mutant proteins was characterized with respect to its kinetic parameters at pH 7 and the pH dependence of ki...

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Bibliographic Details
Published in:Biochemistry (Easton) 1999-08, Vol.38 (35), p.11231-11238
Main Authors: Zhang, Lei, Chooback, Lilian, Cook, Paul F
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution - genetics
Animals
Catalysis
Deuterium - chemistry
Hydrogen-Ion Concentration
Kinetics
Liver - enzymology
Lysine - chemistry
Lysine - genetics
Lysine - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphogluconate Dehydrogenase - chemistry
Phosphogluconate Dehydrogenase - genetics
Phosphogluconate Dehydrogenase - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sheep
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Summary:Site-directed mutagenesis was used to change K183 of sheep liver 6-phosphogluconate dehydrogenase to A, E, H, C, Q, R, and M to probe its possible role as a general base catalyst. Each of the mutant proteins was characterized with respect to its kinetic parameters at pH 7 and the pH dependence of kinetic parameters for the K183R mutant enzyme. The only mutant enzyme that gives a significant amount of catalysis is the K183R mutant, and the extent of catalysis is decreased by about 3 orders of magnitude; the general base pK is perturbed to a pH value of >9. All other mutant enzymes exhibit rates that are decreased by about 4 orders of magnitude compared to that of the wild-type enzyme. Data are consistent with the general base function of K183.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi990433i