Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 2. Site-directed mutagenesis of an essential arginine contained within an apparent P-loop.
Pyruvate phosphate dikinase catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The Arg 561 residue of Clostridium symbiosum PPDK is contained within a Gly-rich stretch of sequence spanning positions 553-563 (viz., GAEGIGLCRTE) located in the 35 kDa C-terminal domain of the enzyme. The possible role of this stretch of sequence as a phosphate binding loop participating in catalysis of the PEP/pyruvate partial reaction (viz., E+PEP<-->E-P+pyruvate, where E-P represents enzyme phosphorylated at the catalytic histidine) was deduced from the similarity of this sequence to other known phosphate binding loops and by its location in the 35 kDa PEP/pyruvate binding domain of PPDK. To test the proposed role of Arg 561, and hence, the signature sequence, in catalysis of the E+PEP<-->E-P+pyruvate partial reaction, the C. symbiosum PPDK site-directed mutants Arg 561-->Leu 561 and Arg 561-->Lys 561 were constructed and expressed in Escherichia coli JM101. Neither mutant catalyzed the full PPDK reaction, ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP, but both catalyzed the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction as efficiently as wild-type PPDK. Both mutants were shown to be unable to catalyze the PEP/pyruvate partial reaction. On the basis of these results it was proposed that Arg 561 and, possibly, the Gly-rich stretch of sequence spanning positions 553-563 are essential components of the active site of the PEP/pyruvate partial reaction.
Yankie
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Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Xu
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Dunaway-Mariano
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D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D016384:Consensus Sequence; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D001120:Arginine; D011772:Pyruvate, Orthophosphate Dikinase; D003545:Cysteine