Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

Journal Article · Tue Jan 26 04:00:00 EST 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00402a020· OSTI ID:7000110

Wang, H C; Ciskanik, L; Dunaway-Mariano, D; von der Saal, W; Villafranca, J J

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze (/sup 14/C)pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, (/sup 14/C)AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and (/sup 32/P)Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using (alpha beta-18O, beta, beta-/sup 18/O/sub 2/)ATP and (beta gamma-18O, gamma, gamma, gamma-/sup 18/O/sub 3/)ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.

• Research Organization: Univ. of Maryland, College Park (USA)
• OSTI ID: 7000110
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:2; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMP
ATP
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS 31
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RADIOISOTOPES
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
TRACER TECHNIQUES
TRANSFERASES