12 - substrates motif

Wu J, Ma QN, Lam KS, Identifying substrate motifs of protein kinases by a random library approach, Biochemistry, 33 14825-14833, 1994. 

Obata T, et al. Peptide and protein library screening defines optimal substrate motifs for AKT/PKB. J. Biol. Chem. 2000 275 36108-36115. 

FIGURE 39.3 Enzyme-sensitive materials incorporating protease substrate motife. 

Fig. 8. Autoradiograms of a model library of structure R-Xj-S-Xj-M-TentaGel incubated with 7[ P] ATP either with (Panel A) or without (Panel B) the catalytic subunit of protein kinase A (PKA-C). Only the first four Edman cycles were performed for this experiment. Side-by-side spreads of the beads and exposure on a single film for 3 h at RT are shown. The sequences of labeled beads are indicated along with the percentage of the total radiolabel competed off by nonradioactive ATP. It is clear that considerable amounts of label can associate noncovalently with diverse sequences, so competition and quantitative methods are important to successful screening. The substrate motif for PKA-C is RRXS (13). represents a sequencing cycle with a proprietary unnatural amino acid.

Figure 23.12 Comparison of the first transition-state mimetics based on the Asn-Phe-Pro substrate motif.

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

The Pictet-Spengler reaction is one of the key methods for construction of the isoquinoline skeleton, an important heterocyclic motif found in numerous bioactive natural products. This reaction involves the condensation of a P-arylethyl amine 1 with an aldehyde, ketone, or 1,2-dicarbonyl compound 2 to give the corresponding tetrahydroisoquinoline 3. These reactions are generally catalyzed by protic or Lewis acids, although numerous thermally-mediated examples are found in the literature. Aromatic compounds containing electron-donating substituents are the most reactive substrates for this reaction. 

Insulin Receptor. Figure 1 Structure and function of the insulin receptor. Binding of insulin to the a-subunits (yellow) leads to activation of the intracellular tyrosine kinase ((3-subunit) by autophosphorylation. The insulin receptor substrates (IRS) bind via a phospho-tyrosine binding domain to phosphorylated tyrosine residues in the juxtamembrane domain of the (3-subunit. The receptor tyrosine kinase then phosphorylates specific tyrosine motifs (YMxM) within the IRS. These tyrosine phosphorylated motifs serve as docking sites for some adaptor proteins with SRC homology 2 (SH2) domains like the regulatory subunit of PI 3-kinase. 

Ubiquitin modification of substrates can be sensed by proteins, which serve as ubiquitin receptors. These proteins harbor domains capable of ubiquitin binding and help to translate the signal into the proper physiological response by forming signaling complexes or activating downstream effectors. So far more than 15 different ubiquitin recognition motifs have been identified. 

HSFl phosphorylation must be sensitive to nonheat inducers of HSF-DNA binding activity because HSFl phosphorylation can be achieved at 37 °C by other inducers of the HS response. HSF 1 contains polypeptide sequences that could serve as substrates for well characterized protein kinases, but few of these are known to be heat inducible. One family of protein kinases, the S6 protein kinases, have already been shown to exhibit heat inducible activity however, their peak level of activity during HS occurs well after the maximal induction of HSF phosphorylation (Jurivich et al., 1991). Thus, other protein kinases are likely to be directly linked to the phosphorylation of HSF. Some of the putative protein phosphorylation sites on HSF include motifs for protein kinase C, casein kinase, and enterokinase. There are tyrosine sequences that match substrates for known tyrosine kinases, but whether these residues are accessible to phosphorylation is not established. 

Upon mutagenesis of the monoamine oxidase from Aspergillus niger (MAO-N) within several rounds of directed evolution [65], variant biocatalysts were identified with largely expanded substrate acceptance, enabling also the deracemization of tertiary amines incorporating straight-chain and cyclic structural motifs [66]. 

---