Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants.
Previous studies had indicated that pyruvate phosphate dikinase (PPDK), an enzyme which catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP), is made up of 25, 13, 18, and 35 kDa domains [Carroll, L. J., Xu, Y., Thrall, S. H., Martin, B. M. & Dunaway-Mariano, D. (1994) Biochemistry 33, 1134]. The catalytic histidine (which mediates the phosphoryl group transfers from ATP to P(i) and pyruvate) is located on the 18 kDa domain while the 25 and 13 kDa domains appear to contain the ATP binding site and the 35 kDa domain appears to contain the pyruvate binding site, respectively. The goal of this investigation was to examine functional interdependency of the putative ATP and pyruvate binding domains. Two truncated forms of PPDK were created by using recombinant DNA techniques. The 35 kDa (C-terminal) deletion mutant was found to catalyze the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction but not the E-P+pyruvate<-->E+PEP partial reaction. The 25 kDa (N-terminal) deletion mutant was found to catalyze the E-P+pyruvate<-->E+PEP partial reaction but not the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction. Neither mutant catalyzes the full ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP reaction. These results are interpreted to mean that the ATP and pyruvate binding domains in PPDK are functionally independent, thus providing evidence for separate active sites for catalysis of the two partial reactions.
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Y
Y
Xu
M
M
McGuire
D
D
Dunaway-Mariano
B M
BM
Martin
D001483:Base Sequence; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011994:Recombinant Proteins; D011772:Pyruvate, Orthophosphate Dikinase