A peptide catalyst can replace an essential enzyme in a eukaryotic cell

Abstract Protein enzymes are central to modern biology, yet how catalysis emerged before the evolution of large, folded proteins remains unresolved. Here we show that a short, genetically encoded peptide can replace an essential enzyme in a living eukaryotic cell. We designed minimal peptides containing a Cys-Xaa-Cys catalytic motif and an endoplasmic reticulum retention signal, and identified variants that rescue the otherwise lethal deletion of protein disulfide isomerase (PDI) in Saccharomyces cerevisiae . Cells relying on these peptides remain viable, though they grow more slowly and adapt by activating stress-response pathways, consistent with PDI being replaced by catalysts of lower intrinsic efficiency. Biochemical analyses show that peptide activity depends on local chemical environment and secondary structure rather than a globular fold. These results demonstrate that short peptides can replace an essential cellular reaction in vivo at the system level, supporting the plausibility of peptide-based catalysis as a precursor to modern protein enzymes.