Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis.
The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) was examined using transient kinetic techniques. Single-turnover experiments with [gamma-32P]ATP or [14C]ATP and PPDK were carried out in the presence and absence of Pi to test for pyrophosphorylenzyme and AMP formation, respectively. Formation of the E-PP.AMP complex was found to be followed by Pi binding and the formation of the E-P.AMP.PPi complex. The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+ > Co2+ > Mg2+). Single-turnover experiments with [32P]PEP and PPDK were carried out in the presence and absence of PPi and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction. Phosphorylenzyme formed from the reaction of the E.PEP complex was converted in the presence of AMP and PPi to free enzyme at a rate exceeding the steady-state turnover rate. The reaction sequence for pyruvate phosphate dikinase was determined to be [formula see text] 31P NMR analysis of the phosphorylenzyme in the native (-4.0 ppm) and denatured form (-3.9 ppm) revealed a 3-N-phosphohistidine residue. Complexation of Mg2+ resulted in a 0.3 ppm upfield shift of the phosphorus resonance from native phosphorylenzyme while Mn2+ complexation lead to extensive line broadening, indicative of metal cofactor binding in close vicinity to the phosphoryl group.(ABSTRACT TRUNCATED AT 250 WORDS)
Thrall
S H
SH
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Mehl
A F
AF
Carroll
L J
LJ
Dunaway-Mariano
D
D
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent; D003013:Clostridium / Q000201:enzymology; D003035:Cobalt / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011489:Protein Denaturation; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D011994:Recombinant Proteins / Q000378:metabolism
D002413:Cations, Divalent; D010710:Phosphates; D011773:Pyruvates; D011994:Recombinant Proteins; D003035:Cobalt; D000249:Adenosine Monophosphate; D008345:Manganese; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D011772:Pyruvate, Orthophosphate Dikinase; D008274:Magnesium