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+/-0.25 nM. (13)C-NMR has been used to show that in aqueous media, Z-Phe-[2-(13)C]Ala-glyoxal gives signals at 207.7 p.p.m. and 96.3 p.p.m. showing that both the alpha-keto carbon and its hydrate are present. When this inhibitor is bound to papain a single signal at 209.7 p.p.m. is observed due to the (13)C-enriched carbon. This demonstrates that the glyoxal alpha-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-(13)C]Ala-glyoxal has also been synthesized and its aldehyde carbon is fully hydrated in aqueous solution giving signals at 88.7 p.p.m. and 90.2 p.p.m. when the alpha-keto carbon and its hydrate are present respectively. When this inhibitor is bound to papain a single signal at 71.04 p.p.m. was observed due to the (13)C-enriched carbon showing that the (13)C-enriched aldehyde carbon forms a thiohemiacetal with Cys-25.
Lowther
Jonathan
J
Department of Biochemistry and Centre for Synthesis and Chemical Biology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
Djurdjevic-Pahl
Aleksandra
A
Hewage
Chandralal
C
Malthouse
J Paul G
JP
D000409:Alanine / Q000737:chemistry; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D006037:Glyoxal / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D010206:Papain / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004151:Dipeptides; D013438:Sulfhydryl Compounds; D010649:Phenylalanine; D006037:Glyoxal; D010206:Papain; D003545:Cysteine; D000409:Alanine