Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase.
It has been proposed that "Glu238" within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k(cat) of Arabidopsis thaliana PDK indicates that ionizable groups with pK values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic pK is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K(m,ATP) values. The acidic pK value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same pK values as wild-type AtPDK, contrary to the previous proposal of a "Glu-polarizing" His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.
Tovar-Méndez
Alejandro
A
Department of Biochemistry, University of Missouri, 117 Schweitzer Hall, Columbia, MO 65211, USA.
Hirani
Tripty A
TA
Miernyk
Jan A
JA
Randall
Douglas D
DD
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases