Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

Abstract

The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico

“…Interacting residues (Asn69 and His463) are situated near each other in the 3D structure of human muscle pyruvate kinase. Here we should state that both Asn69 and His463 (see Figure 4) form allosteric binding site for proline and alanine along with Arg42, Asn43, Arg105, Tyr465, Ile468, Phe469 and Pro470 [11,30]. As one can see in Figure 4 A and Figure 4 B, Docking Server included in the area for potential binding sites search the whole beta-bundle of pyruvate kinase and some parts of the protein situated around the beta-bundle.…”

“…As one can see in Figure 4 A and Figure 4 B, Docking Server included in the area for potential binding sites search the whole beta-bundle of pyruvate kinase and some parts of the protein situated around the beta-bundle. Putative site for ethanol binding is situated under the beta-bundle of pyruvate kinase, as well as the known allosteric site for proline and alanine binding [11,30].…”

“…It is likely that ethanol may inhibit activity of muscle pyruvate kinase by the way of binding to the allosteric site which is known to be involved in interaction with several relatively small molecules, such as alanine, proline and phenylalanine [11,30].…”

“…Asn69 and His463 participate in polar interactions with the allosteric inhibitor. So, Docking Server recognized five from nine residues of the described allosteric site [11,30]. Interestingly, alanine was placed to the substrate-binding site (upon the beta-bundle and not under it) at a frequency of 90% with higher estimated free energy of binding equal to -4.88 kcal/mol.…”

Chemo- and regioselective modification of D,L-phenylalanine with α-cyanoacetylenic alcohols in water

“…2 Among them there are ACE-NEP, 3 urokinase 4 and pyruvate kinase inhibitors. 5 Modifications of phenylalanine are active against the infections caused by Gram-positive bacteria (e.g. vancomycin 6 ).…”