Role of Heterogeneous Enzyme Activity in the Formation of Calcium Phosphate Nanomaterials

The enzymatic formation of calcium phosphate (CaP) in bone is a prime example of how living organisms utilize enzymes to precisely regulate biomineral nucleation and growth, serving as inspiration for the design of biomimetic nanomaterials. In this study, we investigated the formation mechanisms of enzymatic CaP in buffered solutions using single molecule enzymology and cryo-transmission electron microscopy to probe the relationship between tissue nonspecific alkaline phosphatase (TNALP) activity and CaP formation pathways. We characterized the dynamics of heterogeneous TNALP activity, which consists of singly and doubly active enzyme molecules during enzymatic CaP formation and identified two distinct formation pathways that produce amorphous calcium phosphate nanoparticles and octacalcium phosphate nanoparticles. These results show the complex and potentially powerful relationship between heterogeneous enzyme activity and CaP formation mechanisms, offering a new level of control in space and time for the design of biomimetic nanomaterials.