A new kinetic diagnostic for enzymatic mechanisms using alternative substrates.

When the concentration of substrate A is varied against fixed and saturating concentrations of alternative substrates B, B', B", etc., and B is similarly varied against alternatives of A, unique pairs of kinetic patterns are generated for each of the major classes of kinetic mechanism for enzymatic catalysis involving two substrates. Thus, a determination of mechanism may be obtained from just two kinetic patterns. The diagnostic has the following advantages over previous usages of alternative substrates and the traditional approach using initial velocity patterns: efficiency (fewer assays are needed), accuracy (large changes in slopes and intercepts are possible), and precision of assays (one substrate is always saturating).