Teaching coupled reactions in the lab class

Abstract

We present some preliminary protocols for coupled reaction assays. Students are asked to move from direct activity assays using alkaline phosphatase (AP) and para‐nitrophenolphosphate into coupled reaction systems. We study phosphofructokinase (PFK) with a coupled reaction assay. To segue from AP to PFK, we spend one lab period trying to force AP to catalyze the reverse reaction and discuss energetics and Le Chatelier’s principle. The concept of reversibility of a reaction and the role of the enzyme as a catalyst for both the forward and reverse reaction is discussed. This lays the groundwork for using the popular pyruvate kinase, lactate dehydrogenase coupled reaction system for measuring the rate of ATP hydrolysis for many enzyme systems including that of our final project, PFK.