A synthetic functional metabolic compartment. The role of propinquity in a linked pair of immobilized enzymes.
A system was created to model the influence of microcompartments on linked enzymatic reactions. Creatine kinase and hexokinase were covalently attached to Sepharose beads. The gel could be perfused in a specially constructed chamber inside a 360-MHz NMR spectrometer at different flow rates with solutions containing various concentrations of substrates. 31P NMR studies were carried out on the linked enzymatic reaction, creatine phosphate + glucose----creatine + glucose 6-phosphate in two enzyme gels differing in only one aspect, the average distance between hexokinase and creatine kinase. At a distance on the order of 0.1 mm between the enzymes, the average bulk concentrations of substrates and products in the perfusate determined the overall function of the linked system. At an average distance of the order of 10 nm, flux through the linked pair was much higher and much less dependent on the concentration of the intermediate substrate/product ADP/ATP. Even at adenine nucleotide concentrations far below the Km of hexokinase, substantial amounts of glucose 6-phosphate were produced when the enzymes were near but not when they were distant. From saturation transfer measurements and turnover calculations, the lifetime of ATP in the system is estimated to be 0.14-0.5 s when the enzymes are near. This compares to 6 s for distant enzymes. From this it appears that the pair of linked enzymes comprise a functional compartment supported by propinquity in which hexokinase has preferential access to ATP produced by creatine kinase, and creatine kinase to ADP from the hexokinase reaction.
Fossel
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Department of Radiology, Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts 02215.
Hoefeler
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D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012685:Sepharose
D004800:Enzymes, Immobilized; D012685:Sepharose; D006593:Hexokinase; D003402:Creatine Kinase