Assessment of the Enzymatic Dephosphorylation Kinetics in the Assemblies of a Phosphopentapeptide that Forms Intranuclear Nanoribbons

Abstract

Although the formation of peptide assemblies catalyzed by alkaline phosphatase (ALP) has received increasing attention in inhibiting cancer cells, the detailed enzyme kinetics of the dephosphorylation of the corresponding phosphopeptide assemblies have yet to be determined. We recently discovered that assemblies from a phosphopentapeptide can form intracellular nanoribbons that kill induced pluripotent stem cells or osteosarcoma cells, but the kinetics of enzymatic dephosphorylation remain unknown. Thus, we chose to examine the enzyme kinetics of the dephosphorylation of the phosphopentapeptide [NBD-LLLLpY (1)] from concentrations below to above its critical micelle concentration (CMC). Our results show that the phosphopeptide exhibits a CMC of 75 μM in phosphate saline buffer, and the apparent V max and Km values of alkaline phosphatase catalyzed dephosphorylation are approximately 0.24 μM/s and 5.67 mM, respectively. Despite dephosphorylation remaining incomplete at 60 min in all the concentrations tested, dephosphorylation of the phosphopeptide at concentrations of 200 μM or above mainly results in nanoribbons, dephosphorylation at concentrations of CMC largely produces nanofibers, and dephosphorylation below the CMC largely generates nanoparticles. Moreover, the formation of nanoribbons correlates with the intranuclear accumulation of the pentapeptide. By providing the first examination of the enzymatic kinetics of phosphopeptide assemblies, this work further supports the notion that the assemblies of phosphopentapeptides can act as a new functional entity for controlling cell fates.

Enzymatic control of intermolecular interactions for generating synthetic nanoarchitectures in cellular environment

“…To further explore the potential of enzymatic reaction for creating cellular nanostructures, several limitations remain to be addressed, such as enzyme kinetics [ 70 ], dynamics of enzyme locations, and molecular structures of the nanoarchitectures. The dynamic nature of molecular assemblies in vivo presents a challenge in determining enzyme kinetics in cellular environments, especially when multiple enzymes act on the same substrates, or multiple substrates can be transformed by the same enzymes.…”