Single molecules of highly purified bacterial alkaline phosphatase have identical activity

The central paradigm of chemistry is that molecular structure determines mo lecular function. Details of this paradigm can be tested with single-molecu le enzymology, where the activity of individual molecules is studied. In al l cases reported thus far, there is a large molecule-to-molecule heterogene ity in activity and activation energy. This heterogeneity must arise from d ifferences in structure. Replicate incubations on the same molecule yield c onsistent results; the structural heterogeneity must be stable over the tim e period of the experiment, which can extend over several hours. In this pa per, we demonstrate that highly purified molecules of bacterial alkaline ph osphatase generate identical activity; structurally identical molecules beh ave identically. In contrast, the glycosylated mammalian enzyme demonstrate s a complex isoelectric focusing pattern and has a dramatic molecule-to-mol ecule variation in activity and activation energy. Glycosylation affects bo th the kinetics and energetics of this enzymatically catalyzed reaction.