SPECIFICITY OF TRYPSIN DIGESTION AND CONFORMATIONAL FLEXIBILITY AT DIFFERENT SITES OF UNFOLDED LYSOZYME

Fourteen tryptic peptides and nine intermediates were identified as pr oducts of trypsin digestion of reduced and S-3-(trimethylated amino) p ropylated lysozyme. Kinetics of the appearance and disappearance of th ese products were observed by monitoring the peak areas on the chromat ogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple de cay curves with a single rate constant. In this paper, the trypsin sus ceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 n M trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high sus ceptibility ( 10-20 h(-1)) and the other a low susceptibility (1.0-2.0 h(-1)). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; o n the other hand, the C-terminal half of the polypeptide chain is flex ible enough to fit into the active site of trypsin. In addition, six k inds of pentapeptides were synthesized as analogues of lysozyme fragme nts including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kine tics of tryptic digestion of them were observed. Both k(cat) and K-M w ere determined for these synthetic pentapeptides. The susceptibility o f each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher w hen the susceptible peptide bond is included in proteins than in penta peptides, so long as the conformation of peptide chain is flexible. Ho wever, susceptibilities of a few sites in proteins are lower than thos e in pentapeptides. This means that the peptide chains tend to fold lo cally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but th e site of Lys 33 is not so much, although the hydrolytic rate at Lys 3 3 itself is extremely slow. (C) 1994 John Wiley & Sons, Inc.