Manuscript Accepted to PNAS

The first enzymes emerged ∼4 billion years ago and have subsequently become the most diverse and functionally important component of life. But what were the first enzymes doing and how did they look? We probed the properties of the first enzymes by analyzing phospho-ligand binding across all known protein evolutionary lineages. We find that phospho-ligand binding was the founding function of the most ancient enzymes. As opposed to younger evolutionary lineages, ancient enzymes preferentially use N termini of α-helices to bind phosphate moieties. The dominance of N-helix binding sites in the earliest enzymes reflects the ability of the α-helix to realize binding via short and simple sequences, including serines and threonines that interact via both the backbone and side chain.