Single molecules of highly purified bacterial alkaline phosphatase have identical activity

The central paradigm of chemistry is that molecular structure determines molecular function. Details of this paradigm can be tested with single-molecule enzymology, where the activity of individual molecules is studied. In all cases reported thus far, there is a large molecule-to-molecule heterogeneity in activity and activation energy. This heterogeneity must arise from differences in structure. Replicate incubations on the same molecule yield consistent results; the structural heterogeneity must be stable over the time period of the experiment, which can extend over several hours. In this paper, we demonstrate that highly purified molecules of bacterial alkaline phosphatase generate identical activity; structurally identical molecules behave identically. In contrast, the glycosylated mammalian enzyme demonstrates a complex isoelectric focusing pattern and has a dramatic molecule-to-molecule variation in activity and activation energy. Glycosylation affects both the kinetics and energetics of this enzymatically catalyzed reaction.