Cleavage preference of metalloprotease EA1

The cleavage preference of the thermophilic metalloprotease from Geobacillus sp. EA1 was characterised using mass spectrometry, confirming its similarity to thermolysin in preferring P1’ hydrophobic amino acids. While EA1 showed similar efficiencies for cleavage of short peptides with one of six hydrophobic amino acids at the P1’ position, thermolysin showed marked preference for leucine. A single amino acid difference in the S1’ pocket of these similar enzymes provides a rationale for this difference. A variant of EA1 (F133L) had intermediary preference, suggesting that approximately half the difference in preference is attributable to this single amino acid change. Broader preference and higher efficiency make EA1 a superior candidate for quick digestion of varied protein substrates.