Horse Urinary Kallikrein, II. Effect of Subsite Interactions on its Catalytic Activity

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Abstract
The effect of secondary-subsite interactions on the catalytic efficency of horse urinary kallikrein was studied using as substrates oligopeptides and peptidyl-4-nitroanilides with L-Arg at P1. The known secondary specificity of tissue kallikreins for hydrophobic residues at P2 was also demonstrated for horse urinary kallikrein and a higher preferences of this enzyme for L-Phe over L-Leu at P2 was evident. Interaction of subsites S3 with D-Pro and D-Phe enhanced the catalytic efficiency but tripeptidyl-4-nitroanilides with acetyl-D-Pro, L-Pro and acetyl-L-Pro at p3 were no better substrates than acetyldipeptidyl-4-nitroanilides. The importance of the leaving group for the catalysis was proved by higher kcat/Km values for the peptides in relation to peptidyl-4-nitroanilides containing a common acyl-chain. The low kcat value for the peptide with L-Pro at P''2 stresses the importance of a hydrogen bond between P''2 amide and the carbonyl group at S''2. One L-arginine residue at the leaving group, specially at the P''2 position, decreases the value of the apparent km. This effect resulting of side-chain interactions with S''2, is impaired by a second L-Arg at P''1.