The best variant in which the two amino acids at that cleavage site were replaced by two arginine residues was five times more stable than the wild-type enzyme. increased the stability of Pseudomonas glumae lipase to subtilisin by introduction of charged amino acid or proline residues near the primary proteolytic cleavage site ( Frenken et al., 1993). While for proteinase K the Ala20–Ser21 peptide bond was also identified as the primary cleavage site ( Rauber et al., 1978), elastase was reported to cleave the neighbored Ala19–Ala20 peptide bond ( Klee, 1965).Īlthough protein engineering enables tailoring of many structural properties of proteins, the improvement of proteolytic resistance has rarely been the goal of directed or random mutagenesis. A similar fragmentation of RNase A was found by the likewise unspecific proteases proteinase K and elastase.
The fragments comprising the amino acid residues 1–20 (S-peptide) and 21–124 (S-protein) indicate that native RNase A is mainly cleaved between the residues Ala20 and Ser21 which are located in the well accessible loop between two helices (Figure 1 ). The nicked protein which is called ribonuclease S is still active under native conditions but it is separated into two fragments in SDS–PAGE. Although it is characterized by a very compact global tertiary structure, which is stabilized by four disulfide bridges ( Wlodawer et al., 1982), and a high thermodynamic stability ( Pfeil, 1998), it is cleaved by subtilisin Carlsberg even at 25☌ ( Richards and Vithayathil, 1959). An example is the well known bovine pancreatic ribonuclease A (RNase A). Even small, highly compact and thermostable proteins may contain protease-sensitive regions. Unspecific proteases, however, are also able to attack a protein in its native conformation if it contains loop regions that are accessible and flexible enough for a cleavage ( Price and Johnson, 1990 Hubbard, 1998). The reason for this coherence is that most proteins in their native conformation are resistant to proteases, whereas they are rapidly degraded after unfolding. Proteolytic stability generally correlates with the thermal stability of proteins ( McLendon and Radany, 1978 Daniel et al., 1982 Parsell and Sauer, 1989 Akasako et al., 1995). The resistance to proteolytic attack is one of the most important criteria for the industrial application of enzymes. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. Ser21–Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase.
These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS–PAGE, decreased by two orders of magnitude. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2′,3′-cCMP and in its thermodynamic stability.
PROTEOLYTIC FRAGMENT DEFINITION PRO
With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20.
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