Isoleucine residues

As noted by the original authors (Dorovska et al., 1972), and cited by Fersht (1985), there is an excellent linear correlation between log/ccat/KM and the Hansch hydrophobicity parameters (v) of the side chains (Fig. 9, A), except for the two branched side chains (valine and isoleucine residues). However, since the ku values for the esters do vary somewhat (Table A6.8), the values of pKrs do not correlate as strongly with ir (Fig. 9, B). Moreover, the plot shows distinct curvature which probably indicates the onset of a saturation effect due to the physical limits of the Sj binding pocket, adjacent to the enzyme s active site. Still, the points for valine and isoleucine deviate below the others, suggesting that the pocket has a relatively narrow opening. 

Fig. Z14. The activation of chymotrypsin via proteolytic cleavage, a) Chymotrypsinogen is transformed into the active forms of chymotrypsin n and a by trypsin and autoproteolysis, b) The N-terminal isoleucine residue Ile6 is particularly important for the activity of chymotrypsin. The positively charge NH2 group of llel6 interacts electrostatically with Aspl94 and stabilizes an active conformation of the catalytic center. After Stryer Biochemistry , with permission.

The neighboring sequences of the phosphotyrosine residue are decisive for binding specificity of a SH2 domain. At this point, the structure shows that particularly the isoleucine residue at position +3 relative to phosphotyrosine is bound in a very specific manner in a pocket of the SH2 domain (Fig. 8.11). Binding of the peptide to the SH2 domain in Src kinase has therefore been compared to binding of a two-pole plug in a complementary socket, where one of the poles is phosphotyrosine and the other is the amino acid at position +3. 

Fig. 8.11. Recognition of phosphotyrosine-containing substrate peptides by SH2 domains of Src kinase and phospholipase Cyl. Binding of phosphotyrosine-containing peptides to SH2 is shown schematically, based on crystal structures of the complexes. The SH2 domain of Src kinase has a basic binding pocket for the phosphotyrosine residue and a hydrophobic pocket for the isoleucine residues at position -1-3 of the peptide substrate. The SH2 domain of PL-Cyl has a hydrophobic binding surface to which the C-terminal part of the peptide P-Tyr-Ile-Ile-Pro-Leu-Pro-Asp binds. According to Cohen, (1995).

It has been known for many years, however, that the (3-branched amino acids, especially valine and isoleucine, cause problems in synthesis,14,5] and special care and additional reaction time are required when -substituted amino acids are added to a growing peptide chain in synthesis. For example, in the synthesis of [2,4-diisoleucine]oxytocin efforts to couple the isoleucine to isoleucine by the azide method failed and only the rearranged product was obtained 61 Also, it is much more difficult to hydrolyze peptide bonds formed between two or more contiguous -substituted amino adds using standard 6M HC1 conditions. For example, in the hydrolysis of [2,4-diisoleucine]oxytocin (3 isoleucine residues adjacent to each other) complete hydrolysis takes 60 hours. 

A functional NES comprised of hydrophobic leucine and isoleucine residues has been identified in the IVR domain of Keapl [Velichkova and Hasson, 2005], Mutation of the hydrophobic residues as well as leptomycin B (LMB) treatment, which inactivates Crml/exportin, resulted in nuclear accumulation of both Nrf2 and Keapl. It was further demonstrated that the NES in Keapl is required for termination of Nrf2/ARE signaling, and the Nrf2/Keapl complex does not bind to the ARE [Sun et al., 2007], Conversely, in another report the subcellular distribution of endogenous Keapl was essentially similar before and after LMB treatment [Watai et al., 2007], This discrepancy could possibly be... 

Fig. 5.6 Part of the pseudoreceptor model for histamine H3-receptor antagonists [25] (one leucine beside the isoleucine residue is omitted for clarity). Hydrogen bonds from ligands to complementary functions of asparagine (Asn), tyrosine (Tyr) and aspartate (Asp) are located in the GR/D-...

For the 1H/I5N experiment, uniformly labeled substance is obtainable at a reasonable cost, but uniform 13C labeled proteins are more expensive. However, in some cases selective labeling of the methyl groups in valine, leucine, or isoleucine residues of a protein proves sufficient for screening purposes (59). The employment of HSQC experiments to detect binding is especially well exemplified in the technique termed SAR by NMR (33). 

The high level of conservation of tyrosine and lysine residues suggests an important role for these amino acids and their post-translational derivatives in both adhesive and cohesive processes. As mentioned earlier, Waite has proposed that lysine and quinone residues are involved in protein crosslinking. Substitutions at other positions generally result in the presence of serine, threonine, proline (perhaps hydroxyproline), alanine, and isoleucine residues, with an emphasis on polar residues that can interact with most biological surfaces. The resulting protein is rich in the six amino acids tyrosine, lysine, alanine, serine, threonine and proline. Preliminary characterization of cDNA clones encoding... 

Isocitrate dehydrogenase, transhydro-genase and, 52-53, 81, 84, 86-88 Isoleucine residues, disulfide oxidoreduc-tases, 104... 

Figure 12 C-HSQC of PTPIB with ligand (red) and without (black) ligand. The chemical shift change for the isoleucine residue 219 indicates binding of the ligand to the corresponding amino acid...

Figure 18 SOS-NMR using deuterated and selectively protonated protein can resolve ambiguity of ligand orientation, (a) Bottom 1 D H NMR reference spectrum that shows the resonances of the ligand SB203580. Middle 1 D H STD spectrum recorded in the presence of p38a. Top 1D H STD spectrum recorded of a sample prepared according to the SOS procedure of perdeuterated kinase and unlabeled isoleucine residues. A pronounced STD effect for the HI and H4 protons and only weak effects for the H6 and H5 protons resolves ambiguity of ligand orientation in the binding pocket, (b) Distance analysis of the reference X-ray structure.

Supporting evidence for this proposal was obtained from amino acid analyses which demonstrated that glycine was the only typical or non N-methylated amino acid present in 4ab and Sab. Marfey analysis of the acid hydrolysates of 4ab, Sab and majusculamide C (6) confirmed that 4ab and Sab differed from majusculamide C (6) in possessing an N-methyl-L-alanine residue rather than an L-alanine residue. Moreover, 4ab and Sab contained an N-methyl-L-leucine residue rather than an N-methyl-L-isoleucine residue,... 

Active again. A mutation that changes an alanine residue in the interior of a protein to valine is found to lead to a loss of activity. However, activity is regained when a second mutation at a different position changes an isoleucine residue to glycine. How might this second mutation lead to a restoration of activity ... 

Figure 10.30. Binding of Pseudosubstrate to Protein Kinase A. The two arginine side chains of the pseudosubstrate form salt bridges with three glutamate carboxylates. Hydrophobic interactions are also important in the recognition of substrate. The isoleucine residue of the pseudosubstrate is in contact with a pair of leucine residues of the enzyme.

For example, the leader peptide for the phenylalanine operon includes 7 phenylalanine residues among 15 residues. The threonine operon encodes enzymes required for the synthesis of both threonine and isoleucine the leader peptide contains 8 threonine and 4 isoleucine residues in a 16-residue sequence. The leader peptide for the histidine operon includes 7 histidine residues in a row. In each case, low levels of the corresponding charged tRNA causes the ribosome to stall, trapping the nascent mRNA in a state that can form a structure that allows RNA polymerase to read through the attenuator site. 

A resolution details such as identification of carbonyl oxygens allow the conformation of polypeptide to be established with precision. Details of side chains, e.g., forking of isoleucine residues, becomes apparent and in certain cases assignment of amino acid type can be made without sequence data. 

Creamer et al. have shown a strong correlation between decreased helix propensity of amino acid residues and the entropic effects of holding a valine or isoleucine residue in an a-helical conformation (185). This results from the limitation on %1 values of the (3-branched amino acids imposed by the helical backbone conformation. 

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