A new lysine derived glyoxal inhibitor of trypsin, its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin

• Z-Lys-COCHO cyclizes forming a Schiff base.
• The Schiff base forms an enol that breaks down to form an alcohol.
• Z-Lys-COCHO binding to trypsin inhibits this cyclisation reaction.
• Z-Lys-COCHO forms a tetrahedral adduct with trypsin.
• The effective molarity of the trypsin active site serine hydroxyl is ~25,000 M.

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. Ki values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (–COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-13COCHO and Z-Lys-CO13CHO have been synthesized. Using Z-Lys-13COCHO we have observed a signal at 107.4 ppm by 13C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the 13C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO13CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3 ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300 M. At pH 10.3 the free glyoxal inhibitor rapidly (t1/2=0.15 h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t1/2=23 h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.