Understanding mechanisms of enzyme co-operativity: The importance of not being at equilibrium

The discovery at the end of the 1950s and the beginning of the 1960s that there were enzymes like threonine deaminase and aspartate transcarbamoylase that failed to follow the expected hyperbolic behaviour predicted by the Michaelis–Menten equation, raised several questions and induced the development of mechanisms to explain this peculiar behaviour. At that time it was already known that the binding of oxygen to haemoglobin did not follow a hyperbolic curve, but a sigmoidal one, and it was thought that a similar situation probably existed for enzymes with sigmoidal kinetics. In other words, the observed kinetic behaviour was a consequence of co-operativity in the substrate binding. Two main models were postulated: those of Monod, Wyman and Changeux in 1965 and of Koshland, Némethy and Filmer in 1966. Both consider that the different conformations are in equilibrium and that there is a rapid equilibrium in the binding, which implies that co-operativity could only exist if there is more than one substrate binding site per enzyme molecule, that is, if the enzyme is an oligomer. What about monomeric enzymes, could they show kinetic co-operativity? Yes, but only through mechanisms that imply the existence of enzyme conformations that are not in equilibrium, and have different kinetic parameters. There are, in fact, very few examples of monomeric enzymes showing kinetic co-operativity with a natural substrate. The case of “glucokinase” (hexokinase D or hexokinase IV), a monomeric enzyme with co-operativity with respect to glucose, will be discussed.