Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis

• Published in: Biochemistry (Easton) Vol. 32; no. 7; pp. 1803 - 1809
• Main Authors: Thrall, Sara H, Mehl, Andrew F, Carroll, Lawrence J, Dunaway-Mariano, Debra
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23-02-1993
• Subjects: Adenosine Monophosphate - metabolism; Adenosine Triphosphate - metabolism; Bacteriology; Biological and medical sciences; Catalysis; Cations, Divalent; characterization; Clostridium - enzymology; Clostridium symbiosum; Cobalt - pharmacology; covalent; enzymes; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; intermediates; Kinetics; Magnesium - pharmacology; Magnetic Resonance Spectroscopy; Manganese - pharmacology; Metabolism. Enzymes; Microbiology; Phosphates - metabolism; Phosphoenolpyruvate - metabolism; Protein Denaturation; Pyruvate, Orthophosphate Dikinase - metabolism; pyruvate, water dikinase; Pyruvates - metabolism; Pyruvic Acid; Recombinant Proteins - metabolism; Reaction intermediate; Enzyme; Clostridiaceae; Analytical method; Clostridiales; Bacteria; NMR spectrometry; Kinetics; Pyruvate,orthophosphate dikinase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00058a014

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.