Kallistatin, a
serpin that specifically inhibits human
tissue kallikrein, was demonstrated to be cleaved at the
Phe-Phe bond in its reactive site loop (RSL) by
cathepsin D. Internally quenched fluorescent
peptides containing the amino acid sequence of
kallistatin RSL were highly susceptible to hydrolysis by
cathepsin D. Surprisingly, these
peptides were efficiently hydrolyzed at
Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for
cathepsin D. Due to the importance of
cathepsin D in several physiological and
pathological processes, we took the
peptide containing
kallistatin RSL sequence, Abz-Ala-Ile-
Lys-Phe-
Phe-Ser-
Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3'
protease subsites (EDDnp=N-[2,4-dinitrophenyl]-
ethylenediamine and Abz=ortho-amino
benzoic acid). We present in this paper some internally quenched fluorescent
peptides that were efficient substrates for
cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-
Ala-Phe-
Phe-Ser-
Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).