Single molecule processivity and dynamics of cAMP-dependent protein kinase (PKA)

Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 $\mu $s resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Statistical analysis of many events determines on- and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.