Glutamate 190 Is a General Acid Catalyst in the 6-Phosphogluconate-Dehydrogenase-Catalyzed Reaction

• Published in: Biochemistry (Easton) 1998-11, Vol.37 (45), p.15691-15697
• Main Authors: Karsten, William E, Chooback, Lilian, Cook, Paul F
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Catalysis; Consensus Sequence; Glutamic Acid - chemistry; Glutamic Acid - genetics; Hydrogen-Ion Concentration; Kinetics; Liver - enzymology; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphogluconate Dehydrogenase - chemistry; Phosphogluconate Dehydrogenase - genetics; Sequence Alignment; Sheep
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9812827

Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K 6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K 6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.