Active site mapping of human and rat urinary kallikreins by peptidyl chloromethyl ketones

The reactivity of human and rat urinary kallikrein has been determined with peptides of arginine and lysine chloromethyl ketone. Pro-Phe-ArgCH 2Cl, the reagent corresponding to the sequence of kininogen hydrolyzed by kallikrein, was considerably more effective than reagents containing other substituents in the P 1 and P 2 positions (the Arg and Phe binding sites, respectively). Pro-Phe-ArgCH 2Cl inactivates the human enzyme at the 10 −5 m level ( K i 45 μ m, k 2 0.36 min −1) and the rat enzyme at the 10 −6 m level ( K i 4.8 μ m, k 2 0.26 min −1). More effective reagents were obtained by substitution of d-Phe for the P 3 Pro and addition of a dansyl residue in the P 4 position, yielding reagents effective at the 10 −7 m level for both kallikreins. Expansion of the sequence of kininogen to accomodate the P 4 and P 5 binding sites of kallikrein resulted in a reagent, Phe-Ser-Pro-Phe-ArgCH 2Cl, which is approximately 6-fold more reactive than the corresponding tripeptide analog for human kallikrein, while for the rat kallikrein, the tri- and pentapeptide analogs are comparable in reactivity. The importance of Arg in the P 1 position and Phe in the P 2 positions in the sequence of kallikrein's physiological substrate in determining specificity was shown by comparison of the reactivities of the proteases with Ala-Phe-ArgCH 2Cl and Ala-Phe-LysCH 2Cl and with Pro-Phe-ArgCH 2Cl and Pro-Gly-ArgCH 2Cl. Substitution of Lys for the P 1 Arg and substitution of Gly for the P 2 Phe decreased the reactivity of the reagent 10- and 150-fold, respectively, for the human kallikrein and 200- and 250-fold, respectively, for the rat kallikrein. Substitution of l-amino acid residues for the P 3 Pro had little effect on the reactivity of human kallikrein with the affinity labels and decreased the reactivity of the rat enzyme with the affinity labels from 3- to 6-fold.