The present study was carried out to determine the energetics of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate (pyr) with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate, and phosphoenolpyruvate (PEP) [E.ATP <==> E-PP.AMP <==> E-PP.AMP.Pi <==> E-P.AMP.PPi <==> E-P.pyr <==> E.PEP where E-PP and E-P represent the pyrophosphoryl and phosphoryl enzyme intermediates]. Thermodynamic techniques were used along with steady-state and pre-steady-state kinetic techniques to determine the rate constants for the substrate/product binding and release steps and the rate constants for the forward and reverse chemical steps. These values were used along with estimates of the cellular concentrations of the substrates and products to construct the free energy profile for the enzymatic reaction under physiological conditions. The energy profile obtained with the Mg2+/NH4(+)-activated enzyme revealed well-balanced transition states and well-balanced internal ground state energies (i.e., within 1 kcal/mol of each other). Examination of the energetics of the reaction steps leading from ATP to phosphohistidine formation in E-P suggested the use of intrinsic binding energy in the synthesis of a high energy P-N linkage. Comparison of the energy profiles of the Mg2+/NH4(+)-vs Co2+/NH4(+)-activated enzymes revealed cofactor selectivity at each of the phosphosphoryl transfer steps.

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Few laboratory procedures describe the use of circular dichroism (CD) at the undergraduate level. To increase the number of laboratory exercises using CD, a thermal denaturation study of myoglobin using CD is described to assess protein stability. Values obtained from a more classic linear data analysis approach are consistent with data analyzed with a modern software program that "fits" the data set to a model using a non-linear approach. Both methods gave quantities consistent with reported literature values.

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)