Comparison of the Kinetics of Cathepsins B, L, L-like, and X from the Dorsal Muscle of Mackerel on the Hydrolysis of Methylcoumarylamide Substrates

Proteolytic kinetics of cathepsins B, L, L-like, and X (a novel cysteine proteinase) from mackerel dorsal muscle were determined using methylcoumarylamide peptides as substrates. From a comparison of K m and K cat / K m of these proteinases, cathepsin X showed a lower K m (0.9 μM) and higher K cat / K m (1216.7 μM - 1 min - 1 ) for the hydrolysis of Z-Arg-Arg-MCA. Hydrolysis of Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA by cathepsin L-like had lower K m (1.5 and 1.3 μM, respectively) and higher K cat / K m (2404.5 and 437.1 μM - 1 min - 1, respectively). Cathepsins L and L-like did not hydrolyze Boc-Glu-Lys-Lys-MCA, Suc-Leu-Leu-Val-Tyr-MCA, and Z-Pyr-Gly-Arg-MCA. Cathepsin B had a higher K m (157.8 μM) and lower K cat / K m (94.1 μM - 1 min - 1 ) for the hydrolysis of Boc-Val-Leu-Lys-MCA. This property could be used to differentiate cathepsin B from cathepsin L or L-like. For the hydrolysis of methylcoumarylamide peptides, the kinetics of cathepsins B, L, L-like, and X were quite different. Peptides with basic amino acids, such as Lys or Arg, at the P 1 site were more susceptible to these proteinases.