A 13C-NMR study of the inhibition of papain by a dipeptide-glyoxal inhibitor

Z-Phe-Ala-glyoxal (where Z is benzyloxycarbonyl) has been synthesized and shown to be a competitive inhibitor of papain with a K_i = 3.30±0.25nM. ¹³C-NMR has been used to show that in aqueous media, Z-Phe-[2-¹³C]Ala-glyoxal gives signals at 207.7p.p.m. and 96.3p.p.m. showing that both the α-keto carbon and its hydrate are present. When this inhibitor is bound to papain a single signal at 209.7p.p.m. is observed due to the ¹³C-enriched carbon. This demonstrates that the glyoxal α-keto carbon is not hydrated when it is bound to papain and that it does not form a thiohemiketal with the thiol group of Cys-25. Z-Phe-[1-¹³C]Ala-glyoxal has also been synthesized and its aldehyde carbon is fully hydrated in aqueous solution giving signals at 88.7p.p.m. and 90.2p.p.m. when the α-keto carbon and its hydrate are present respectively. When this inhibitor is bound to papain a single signal at 71.04p.p.m. was observed due to the ¹³C-enriched carbon showing that the ¹³C-enriched aldehyde carbon forms a thiohemiacetal with Cys-25.