Horse urinary kallikrein, II. Effect of subsite interactions on its catalytic activity.
The effect of secondary-subsite interactions on the catalytic efficiency 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 preference 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 acetyl-dipeptidyl-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.
Araújo-Viel
M S
MS
Departamento de Bioquímica, Escola Paulista de Medicina, São Paulo, Brasil.
Juliano
M A
MA
Oliveira
L
L
Prado
E S
ES
D000818:Animals; D001665:Binding Sites; D006736:Horses; D007610:Kallikreins / Q000302:isolation & purification / Q000652:urine*; D007700:Kinetics; D011485:Protein Binding; D013379:Substrate Specificity
D007610:Kallikreins