Isotope-exchange evidence for an ordered mechanism for rat-liver glucokinase, a monomeric cooperative enzyme.

The order of addition of substrates and release of products in the reaction catalyzed by rat-liver glucokinase has been studied by measurements of isotope exchange. Experiments at chemical equilibrium showed some degree of randomness, but steady-state experiments showed a predominantly ordered process with glucose binding first and glucose 6-phosphate released last. Experiments to trap binary complexes in the steady state demonstrated the existence of complexes of the enzyme with glucose and with glucose 6-phosphate but gave no evidence for the occurrence of corresponding complexes with ATP or ADP. Flux ratios measured in both the forward and reverse reactions provided a more rigorous and quantitative confirmation of these characteristics of the reaction. These observations support the interpretation of glucokinase cooperativity in terms of a "mnemonical" mechanism and conflict with an alternative interpretation in terms of a random addition of substrates.