ACCURATE STEADY-STATE APPROXIMATIONS - IMPLICATIONS FOR KINETICS EXPERIMENTS AND MECHANISM

A theoretical analysis of accurate steady-state experiments is presented. In principle, mechanisms can be distinguished and all rate constants found independently by using this approach. The method is illustrated by comparing two enzyme-catalysis mechanisms originally proposed by Henri: the usual enzyme-complex model, and a bimolecular model. It is well-known that both mechanisms give the hyperbolic rate law in the standard kinetic analysis; therefore, it is believed that steady-state kinetics is unable to distinguish between them. However, we show that the hyperbolic rate law is only the lowest order approximation in a systematic procedure for getting more accurate formulas for the reaction velocity. In higher order approximations the two model mechanisms are (mathematically) distinct: At any substrate concentration, the enzyme-complex mechanism has a larger velocity than the hyperbolic law, and the bimolecular mechanism a smaller velocity. This difference is most obvious in Woolf or Eadie-Hofstee plots where the hyperbolic law is linear, but the two mechanisms give curves with distinctive characteristics. In general, these systematic methods provide very accurate steady-state velocity expressions, and they can be applied to accurate experimental results to yield more kinetic information.