Inhibitors substrate-enzyme interactions

To understand the inhibition of a-amylase by peptide inhibitors it is crucial to first understand the native substrate-enzyme interaction. The active site and the reaction mechanism of a-amylases have been identified from several X-ray structures of human and pig pancreatic amylases in complex with carbohydrate-based inhibitors. The structural aspects of proteinaceous a-amylase inhibition have been reviewed by Payan. The sequence, architecture, and structure of a-amylases from mammals and insects are fairly homologous and mechanistic insights from mammalian enzymes can be used to elucidate inhibitor function with respect to insect enzymes. The architecture of a-amylases comprises three domains. Domain A contains the residues responsible for catalytic activity. It complexes a calcium ion, which is essential to maintain the active structure of the enzyme and the presence of a chloride ion close to the active site is required for activation. 

Competitive, reversible inhibitors bind the active site of the enzyme and therefore block substrate-enzyme interactions. The inhibitor (I) and substrate may not bind simultaneously (Scheme 4.11). In something of a chemical love triangle, the enzyme s binding ability is split between two molecules, the substrate and inhibitor. Therefore, the effective affinity of the enzyme for the substrate alone drops. Km of the substrate will A... 

In spite of the close structural relationship of the molybdenum hydroxylases, including a tendency for hydrophobic substrate/enzyme interaction, there is a very significant difference in the substrate specificity of the two enzymes. Not only is there considerable variation in the affinities for substrates and inhibitors, but there is often a difference in the position of oxidative attack. As both enzymes catalyse apparently similar nucleophilic reactions, this difference cannot be explained solely from electronic considerations and is probably due, to a great extent, to the differential response of each enzyme to steric factors. 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

R. Wolfenden, Conformational Aspects of Inhibitor Design Enzyme-Substrate Interactions in the Transition State , Bioorg Med. Chem. 1999, 7, 647-652... 

The process of reversible inhibition is described by an equilibrium interaction between enzyme and inhibitor. Most inhibition processes can be classified as competitive or noncompetitive, depending on how the inhibitor impairs enzyme action. A competitive inhibitor is usually similar in structure to the substrate and is capable of reversible binding to the enzyme active site. In contrast to the substrate molecule, the inhibitor molecule cannot undergo chemical transformation to a product however, it does interfere with substrate binding. A noncompetitive inhibitor does not bind in the active site of an enzyme but binds at some other region of the enzyme molecule. Upon binding of the noncompetitive inhibitor, the enzyme is reversibly converted to a nonfunctional conformational state, and the substrate, which is fully capable of binding to the active site, is not converted to product. 

Reversible inhibition occurs rapidly in a system which is near its equilibrium point and its extent is dependent on the concentration of enzyme, inhibitor and substrate. It remains constant over the period when the initial reaction velocity studies are performed. In contrast, irreversible inhibition may increase with time. In simple single-substrate enzyme-catalysed reactions there are three main types of inhibition patterns involving reactions following the Michaelis-Menten equation competitive, uncompetitive and non-competitive inhibition. Competitive inhibition occurs when the inhibitor directly competes with the substrate in forming the enzyme complex. Uncompetitive inhibition involves the interaction of the inhibitor with only the enzyme-substrate complex, while non-competitive inhibition occurs when the inhibitor binds to either the enzyme or the enzyme-substrate complex without affecting the binding of the substrate. The kinetic modifications of the Michaelis-Menten equation associated with the various types of inhibition are shown below. The derivation of these equations is shown in Appendix S.S. 

Fig. 15.1 Schematic representation of the binding mode of a substrate in an enzyme to be inhibited (left). Inhibitor 1 (center) and 2 (right) are both competitive inhibitors but their interaction pattern is different, making pharmacophore investigation tricky.

Figure 3. Reaction of a serine protease with a peptide chloromethyl ketone. The side chain of the Pt residue of the inhibitor is shown interacting with the primary substrate binding subsite (SJ of the enzyme.

Wolfenden R (1999) Conformational aspects of inhibitor design enzyme-substrate interactions in the transition state. Bioorg. Med. Chem. 7 647-652... 

The vibrational processes in molecules are also reflected in the Raman spectra (Spiro, 1987, 1988). When the substance is irradiated at a frequency far from the frequency of its absorption, additional (satellite) lines may appear in the scattering light. The origin of such lines is accounted for by the fact that during the interaction of electromagnetic radiation, the molecule part of the radiant energy is transferred to the excited vibrational levels and part of the energy is released from the excited levels. In metalloenzymes and in substrate-enzyme and inhibitor-enzyme complexes the active sites incorporate only a small part of the macromolecular atoms. 

Figure 6 Model for how inhibitors and substrates interact with presenilin. Helical peptides are docking site inhibitors (DSIs) and interact on the outside of the presenilin molecule at the NTF/CTF heterodimeric interface. Transition-state analog inhibitors (TSAs) interact on the inside of the presenilin molecule where the active site resides. The active site, which contains water and two aspartates, is thought to be sequestered away from the hydrophobic environment of the lipid bilayer. These findings have implications for how substrate interacts with the enzyme. The transmembrane domain of the substrate (S) interacts with the docking site and passes either in whole or in part into the active site for proteolysis.

Despite all the problems inherent to QM/CM approaches, some extremely interesting and perceptive work has been described in the literature recently in which all sorts of approaches have been used, improvements introduced and results obtained ([351, 372] and references therein). The study of enzyme catalysed reaction mechanisms, the calculation of relative binding free energies of substrates and inhibitor, and the determination of proton transfer processes in enzymatic reactions, are all good examples of enzyme-ligand interactions studies. Even though Warshel s EVB method [349] probably remains the most practical QM/CM approach for the study of enzyme catalysis, very useful work has been reported on enzyme catalysed reactions ([381] for an excellent review-[238, 319, 382-384]). This is a consequence of the accuracy of QM to treat the active site and inhibitor/substrate and the viability of classical mechanics to model the bulk of the enzyme not directly involved in the chemical reaction. 

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