BIOCHEMICAL THERMODYNAMICS AND ANALYTICAL ENTHALPIMETRY.

摘要

Thermodynamics of enzymatic phosphorylation reactions of the type; Phosphorylated acceptor + Mg(ADO)('-) + H('+) (1);are intercompared critically, encompassing the acceptor/ phosphorylated acceptor pairs: glycerol/glycerol-3-phosphate, pyruvate/phospho(enol)-pyruvate, fructose-6-phosphate/fructose-1,6-diphosphate, glucose/glucose-6-phosphate, and creatine/ phosphocreatine. Heats of reaction were measured by direct injection enthalpimetry (DIE) which is conceptually a quasi-instantaneous thermometric titration. Conditional (DELTA)H('0') values were determined with a precision of two percent at the ninety-five percent confidence level. Standard and reference states were judiciously selected to warrant the approximation that substrates and products which are Bronsted acids or bases existed solely in their most highly charged basic form. Corresponding (DELTA)G('0') and (DELTA)S('0') assignments were evaluated.;The largest influence on enthalpy was found to be relief of electrostatic repulsion. Cleavage and transferral of the terminal phosphate from the highly charged ATP molecule to an uncharged molecule resulted in a large internal energy decrease, as observed in the heats of phosphorylation of glycerol and glucose. Addition of the phosphoryl group to a charge molecule resulted in a lesser internal energy decrease in the fructose phosphate reaction. Entropy changes were accounted for primarily by solvation effects. Enhanced solvation of products relative to reactants resulted in an entropy loss upon phosphorylation of glucose. Preferential solvation of the reactants yielded entropy gains when glycerol, creatine and fructose phosphate were phosphorylated.; Acceptor + Mg(ATO)('2) (DBLHARR);Reaction 1 with glycerol as the acceptor moiety ((DELTA)H('0') = -29.9 kJ (.) mol('-1)) served as the basis for developing a novel approach for the clinically important analysis of triglycerides in human serum. Triglycerides were hydrolyzed with one-hundred percent yield of glycerol. The glycerol generated was phosphorylated in situ and determined by measurement of the heat evolved. Exothermicity was enhanced by coupling Reaction 1 with the proton transfer process; H('+) + RNH(,2) (DBLHARR) RNH(,3)('+); (DELTA)H(,2)('0') = -43.7 kJ/mol (2);where RNH(,2) denotes the buffer tris(hydroxymethyl)aminomethane. The heat corresponding to the sum of ((DELTA)H(,1)('0') + (DELTA)H(,2)('0')) = -73.6 kJ/mol was used as a standard, obviating the need for empirical calibration curves.