Substrate binding contacting residues

Starting at very low temperatures, we know from the pseudophase diagram in Fig. 14.1(a) that the system resides in pseudophase ACT This means that the macrostate of the peptide is dominated by the class of compact, filmlike single-layer conformations. The system obviously prefers surface contacts at the expense of hydrophobic contacts. Nonetheless, the formation of compact hydrophobic domains in the two-dimensional topology is energetically favored, but maximal compactness is hindered by the steric influence of the substrate-binding polar residues. 

Fig. 7.3. Contact points of protein kinase A with inhibitor and peptide substrates. The contact points are shown between the catalytic subunit of protein kinase A, the inhibitor PKI (5 — 24) and a peptide substrate (kemptide). The inhibitor binds, via two Arg residues (P3, P-2), to the same Glu residue of protein kinase A that also binds to the substrate. The phosphorylation site is labelled as P, the A-terminal and C-terminal situated amino acids are labelled as (-) and (+) and are numbered starting from the phosphorylation site. According to Kemp et ah, (1994).

The inhibitor is boimd in an elongated form to the substrate binding surface and forms many contact points to the protein kinase. Hydrophobic residues of the inhibitor find their binding equivalents in hydrophobic pockets of the enzyme. The Arg residues important for specificity of substrate binding are in contact with Glu residues of the... 

Phosphohpase Cy and protein tyrosine phosphatase Syp possess an SH2 domain of class 3. Their substrate binding site has mostly hydrophobic character. The substrate is boimd in a stretched form in a flat pit where contacts are formed to a hydrophobic sequence section of the substrate, including 5—6 amino acids on the C-terminal side of the phosphotyrosine residue. 

Hydration water molecules indicate substrate-binding sites. In crystal structures of native proteins, the active sites are usually hydrated if they are not in direct contact with symmetry-related protein molecules. Since the substrates or inhibitors are recognized by the protein and bound to its active site by hydrogen bonds and/or by insertion of hydrophobic residues into hydrophobic pockets, it is not surprising to find, in the native protein, water associated in positions which are the... 

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.

In cases where there is more than one subunit per asymmetric unit, the lattice contacts may lead to asymmetric binding. In crystals of chicken triose phosphate isomerase, which contain one dimer per asymmetric unit, only one subunit was found to bind stubstrate but there was no half-sites reactivity observed in solution studies. In crystals of the yeast enzyme, where the lattice contacts are different, both subunits bind substrate and undergo substantial conformational change [161]. In both crystal forms there is a loop of chain, residues 168-177, which, in the native enzyme, exhibits conformational flexibility. On binding substrate the loop moves to close the substrate binding site and becomes ordered [223]. In the crystals of the chicken enzyme, movement of this loop for one subunit is blocked by lattice contacts. In the other subunit, movement of the loop and substrate binding are observed, just as in the yeast triose phosphate isomerase crystals. 

Substrate binding involves interactions with both the CDK and the cyclin. Whereas the CDK contacts mainly the residues surrounding the target Ser/Thr of the substrate, interactions between distinct sequence elements of the cyclin and the substrate have been shown to contribute to substrate specificity of cyclin-CDK complexes as well. The region of the cyclin responsible for this interaction is also involved in the binding of CDK inhibitors and other regulatory proteins such as the retinoblastoma protein, pRb (see Section 13.4.2). 

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