12] Partition analysis: Detecting enzyme reaction cycle intermediates

Abstract

Partition analysis leads directly to a reaction rate constant that is specific to a particular intermediate. This is possible, because the concentration of the partitioning intermediate cancels in the expression of the partition ratio and one of the components of the ratio is usually a known steady-state parameter, such as k cat or k cat / K m . This is not to say that the rate constant determined may not be a composite of first-order rate constants, only that it is specific for one intermediate of the reaction cycle. This chapter discusses the subject of partition analysis, as it has evolved since its first application 20 years ago and since a previous review in 1980. The experiments used to describe the methods and concepts are taken from studies with yeast hexokinase and fumarase. With hexokinase, the experiments are related mainly to the processing of the substrates. They are more or less typical of the procedures used to study a wide variety of other enzymes by other workers. With fumarase, it identifies recycling, not product release, as the rate-limiting step and shows how the partition methods may be used to learn how protons travel between solvent and an active site.