Hypotheses for and some kinetic studies with glutamine synthetase and acetate thiokinase

Kinetic studies with glutamine synthetase showed that the apparent Michaelis constant for glutamine was independent of the concentration of ammonia. Similarly, with other substrate combinations little or no interdependency of apparent Michaelis constants was found. Measurements of the rate of isotope exchange at equilibrium with glutamine synthetase showed that by variation of equilibrium conditions the rate of incorporation of glutamate into glutamine could be made considerably less than, equal, to or considerably greater than the rate of incorporation of inorganic phosphate into ATP. Likewise, with acetate thiokinase, variation of equilibrium conditions resulted in a rate of acetate incorporation into acetyl CoA which was approximately equal to or definitely greater than the rate of AMP incorporation into ATP. This demonstrates that with these enzymes, steps other than covalent bond forming and breaking steps can control equilibrium exchange rates. The results, however, limit mechanistic deductions from exchange data. Glutamine synthetase was found to require prior incubation with ATP for demonstration of maximum initial velocity. The significance of the findings are discussed in relation to alternative hypotheses for the glutamine synthetase and acetate thiokinase reactions, including a new hypothesis based on prior reaction of two substrates before reaction with ATP. Present information appears insufficient to allow a choice among various hypotheses.