The P6 glutamic acid is an essential specificity determinant for TEV protease.10,18 As shown in Figure 3, P6 Glu interacts with the loop in between b12 and b13A in TEV protease, whereas the side chain of P6 Glu rotates 90 and factors PA-824 molecular weight mw into the solvent in the TVMV protease peptide cocrystal construction. As shown in Figure 3, the only interaction created by the side chain of P6 Glu from the TVMV peptide framework is actually a hydrogen bond between its Oe1 atom along with the O c atom of P5 Thr. Protease residues make only principal chain contacts with P6 Glu, which ought to have tiny or no effect on sequence specificity. In TEV protease, the interactions with P6 Glu are much more substantial. As shown in Figure 3, atom O e1 and atom O e2 of P6 Glu are inside hydrogen bonding distance of N d2 of Asn171, N d2 of Asn176, and O g of Tyr178. Also, its mainchain O atom makes a hydrogen bond with N d1 of His214. These observations propose the P6 place is often a much more critical specificity determinant for TEV protease than TVMV protease. Constant with this notion, the P6 position in every one of the naturally happening TEV polyprotein processing web pages is occupied by glutamic acid, whereas variations take place with the P6 web pages from the TVMV polyprotein.
10 The side chains of P5 Thr in the TVMV peptide and P5 Asn in TEV peptide each venture away from your proteins into compound library screening solvent.
As a result, consistent using the high diversity of residue forms that come about on the P5 positions with the natural polyprotein processing websites,8 this place really should not be a major specificity determinant for both enzyme. Additionally, biochemical research have shown that practically any residue can occupy the P5 place with tiny or no impact on the cleavage effectiveness of TEV protease.19 The S4 pockets are hydrophobic in each TVMV and TEV proteases. Even so, the S4 pocket of TVMV protease is shallower than that of TEV protease. The Van der Waals cavity volumes in the S4 pockets in TVMV protease and TEV protease are 137 and 241 A ? three, respectively, calculated making use of VOIDOO 20 with a probe radius of one.four A ?. This could make clear why the P4 place is invariably occupied by valine while in the purely natural TVMV processing web pages whereas, despite the fact that leucine would be the residue most regularly found at this place in TEV processing sites, valine and isoleucine also happen in some instances.21 In addition, experiments carried out with oligopeptide substrates in vitro demonstrated that when the P4 valine in an otherwise optimal substrate for TVMV protease was replaced by leucine, it may possibly no lengthier be cleaved through the enzyme.10 That is understandable as the a lot smaller sized S4 pocket of TVMV protease cannot accommodate the bigger side chain of leucine. On the other hand, once the P4 leucine is replaced with valine inside the TEV protease substrate.