Methods of determining the order of addition of substrates and dissociation of products by using flux ratios are investigated. Where an enzyme obeys hyperbolic steady-state velocity kinetics it is concluded that it may be particularly useful to compare the measured flux ratios with those calculated from the steady-state velocity parameters. 2. An expression is derived relating the relative contribution of the two pathways in a branched pathway to the flux ratios. 3. The relationship of equilibrium-reaction-rate measurements [Boyer & Silverstein (1963) Acta Chem. Scand. 17, Suppl. 1, S195] to the flux ratios is considered. Equilibrium-reaction rates are shown to be affected both by the addition of substrates and dissociation of products. Methods of analysing the data to distinguish between these events are discussed. 4. Methods of measurement of flux ratios are described, and it is concluded that the non-equilibrium steady-state method is preferable to measurements at chemical equilibrium. 5. The relative significance of flux ratio measurements and steady-state velocity inhibition data is discussed. It is concluded that flux ratios, when taken in conjunction with the inhibition data, provide the least ambiguous information about mechanism.
