Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase.
The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.
D000255:Adenosine Triphosphate / Q000032:analysis*; D000818:Animals; D004800:Enzymes, Immobilized / Q000494:pharmacology*; D006593:Hexokinase / Q000494:pharmacology; D007313:Insecta / Q000201:enzymology; D007700:Kinetics; D008156:Luciferases / Q000494:pharmacology*; D008433:Mathematics; D008722:Methods; D008832:Microchemistry; D011770:Pyruvate Kinase / Q000494:pharmacology
D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D008156:Luciferases; D006593:Hexokinase; D011770:Pyruvate Kinase