Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 1. Identification of an essential cysteine by chemical modification with [1-14C]bromopyruvate and site-directed mutagenesis.
Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP). The reaction takes place according to the following steps: (1) E+ATP+P(i)<-->E-PP.AMP.P(i), (2) E-PP.AMP.P(i)<-->E-P+AMP+PP(i), and (3) E-P+pyruvate<-->E+PEP, where E represents free enzyme; E-PP, pyrophosphorylenzyme; and E-P, phosphorylenzyme. Steps 1 and 2 comprise the nucleotide partial reaction, and step 3 comprises the pyruvate partial reaction. The present studies were carried out to locate amino acid residues within the primary structure of Clostridium symbiosum PPDK participating in the catalysis of the pyruvate partial reaction. The enzyme was treated with the affinity label [1-14C]bromopyruvate, reduced with NaBH4, proteolyzed with trypsin, and chromatographed on an HPLC column. The radiolabeled tryptic peptide isolate was sequenced to reveal Cys 831 as the site of alkylation. Using PCR techniques Cys 831 was replaced by Ala, and the C831A PPDK mutant formed was then subjected to kinetic analysis. Rapid quench studies of single turnover reactions on the enzyme showed that the mutant is as efficient as wild-type PPDK in catalyzing the nucleotide partial reaction while it is unable to catalyze the pyruvate partial reaction. These results were interpreted as evidence for a role of Cys 831 in pyruvate/PEP binding and/or catalysis.
Xu
Y
Y
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Yankie
L
L
Shen
L
L
Jung
Y S
YS
Mariano
P S
PS
Dunaway-Mariano
D
D
Martin
B M
BM
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin
D000345:Affinity Labels; D001426:Bacterial Proteins; D017931:DNA Primers; D011773:Pyruvates; C017092:bromopyruvate; D010728:Phosphoenolpyruvate; D011772:Pyruvate, Orthophosphate Dikinase; D014357:Trypsin; D003545:Cysteine