Enzyme Interactions

Numerous drugs exert their effects and side effects by interacting with enzymes. Some examples of these interactions are now cited. 

FIGURE 2.3 By inhibiting the synthesis of folic acid, sulfonamides exert their antibacterial activity. 

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Figure 8.2 Enzyme interaction with two enantiomers of a given substrate molecule.

The complex structure of the enzyme can show a very large substrate-enzyme interaction specificity, which can be traduced to a high degree of chemo-, regio-, or stereoselectivity. For this reason, nowadays, the versatility of biotransformations for synthetic proposals is an excellent tool for organic chemists [9]. 

Hannongbua S (2006) Structural Information and Drug-Enzyme Interaction of the Non-Nucleoside Reverse Transcriptase Inhibitors Based on Computational Chemistry Approaches. 4 55-84... 

A. S. Boyd and M. M. Mortland, Enzyme interactions with clays and clay-organic matter complexes. Soil Biochemistry, Vol. 6 (J.-M. Bollag and G. Stotzky, eds.), Marcel Dekker, New York, 1990, p. I. 

The catalytic pi 10 subunit has four isoforms, all of which contain a kinase domain and a Ras interaction site. In addition, the a, (3, and y isoforms possess an interaction site for the p85 subunit. The class I enzymes can be further subdivided class IA enzymes interact through their SH2 domains with phosphotyrosines present on either protein tyrosine kinases or to docking proteins such as insulin-receptor substrates (IRSs GAB-1) or linkers for activation of T cells (LATs in the case of T cells). 

The application of magnetic resonance techniques to biological systems is a relatively new approach for the study of macromolecules. In this review we have presented the different approaches which have been made to study Bi2-enzymes. Clearly some progress has been made particularly from the application of ESR to a study of the enzymes ethanolamine ammonia-lyase and ribonucleotide reductase. Although 13C NMR is well in its developmental stages it is obvious that this technique will prove to be very useful for the examination of coenzyme-enzyme interactions. Studies of how corrinoids bind in enzymes and how sulfhydryl containing proteins are involved in enzyme catalysis comprise two major problems which must be overcome before realistic mechanisms can be presented for this group of enzymes. 

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

The binding of a substrate to its active center was first postulated by E. Fisher in 1894 using the lock and key mechanism which states that the enzyme interacts with its substrate like a lock and a key, respectively, i.e. the substrate has a matching shape to fit into the active site. This theory assumed that the structure of the catalyst was completely rigid and could not explain why the macromolecule was able to catalyze reactions involving large substrates and not those with small ones, or why they could convert non natural compounds with different structural properties to the substrate. 

Extensive studies have established that the catalytic cycle for the reduction of hydroperoxides by horseradish peroxidase is the one depicted in Figure 6 (38). The resting enzyme interacts with the peroxide to form an enzyme-substrate complex that decomposes to alcohol and an iron-oxo complex that is two oxidizing equivalents above the resting state of the enzyme. For catalytic turnover to occur the iron-oxo complex must be reduced. The two electrons are furnished by reducing substrates either by electron transfer from substrate to enzyme or by oxygen transfer from enzyme to substrate. Substrate oxidation by the iron-oxo complex supports continuous hydroperoxide reduction. When either reducing substrate or hydroperoxide is exhausted, the catalytic cycle stops. 

Since the first report on the ferrocene mediated oxidation of glucose by GOx [69], extensive solution-phase studies have been undertaken in an attempt to elucidate the factors controlling the mediator-enzyme interaction. Although the use of solution-phase mediators is not compatible with a membraneless biocatalytic fuel cell, such studies can help elucidate the relationship between enzyme structure, mediator size, structure and mobility, and mediation thermodynamics and kinetics. For example, comprehensive studies on ferrocene and its derivatives [70] and polypy-ridyl complexes of ruthenium and osmium [71, 72] as mediators of GOx have been undertaken. Ferrocenes have come to the fore as mediators to GOx, surpassing many others, because of factors such as their mediation efficiency, stability in the reduced form, pH independent redox potentials, ease of synthesis, and substitutional versatility. Ferrocenes are also of sufficiently small size to diffuse easily to the active site of GOx. However, solution phase mediation can only be used if the future biocatalytic fuel cell... 

Mineral colloid-enzyme interactions have been documented (e.g., Theng 1979 Bums 1986 Naidja et al. 2000 Bums and Dick 2002). Besides cation-exchange reactions, adsorption of enzymes by mineral colloids may proceed through ionic, covalent, hydrophobic, hydrogen bonding, and van der Waals forces. When enzymes are adsorbed on mineral colloids, changes in the tertiary structures (i.e., the folding of the helix or... 

The active site is a specialized region of the protein where the enzyme interacts with the substrate. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

It would appear that the specific action of an enzyme upon its substrate is conditioned by a definite chemical structure and spatial arrangement of the constituent polar and non-polar groups of the enzyme protein as well as by the constitution and configuration of the substrate. In some cases an enzyme interacts with one chemical compound only. For example, galactokinase extracted from Saccharomyces fragilis (grown on whey) catalyzes the transphosphorylation between adenosine triphos-... 

Moazed, D. and Johnson, D. A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Cell, 1996, 86, 667-77. 

To study drug-receptor/enzyme interaction, it is not always convenient or appropriate to use a living system of the target receptor. Instead, biochemical assays can be devised to mimic the target. Very often, the assays use multicolor luminescence or fluorescence-based reagents. In this way, the reaction path can be followed in space and time to enable quantitative evaluation of the reaction. 

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. 

Serine Proteinases. This group of proteinases is the best known because they are more numerous and better characterized than the other three groups. These proteinases are found in virtually all organisms, indicative of their importance and wide ranging proteolytic capabilities. They demonstrate broad substrate specificities with the sites (amino terminal to the scissile bond) generally being more important in enzyme interaction. 

The critical points of the cycle involving substrate-enzyme interactions are illustrated in Figure 7.2 and explored below ... 

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