Enzymic sites, protein environment

Enzymes that catalyze redox reactions are usually large molecules (molecular mass typically in the range 30-300 kDa), and the effects of the protein environment distant from the active site are not always well understood. However, the structures and reactions occurring at their active sites can be characterized by a combination of spectroscopic methods. X-ray crystallography, transient and steady-state solution kinetics, and electrochemistry. Catalytic states of enzyme active sites are usually better defined than active sites on metal surfaces. 

For the present reaction, the presence of surrounding protein only marginally affects the barrier (it increases by 0.7 kcal/mol). A possible reason for the small protein effects could be that in the present model, the active site is not deeply buried inside the enzyme instead it is located on the interface of two monomers. Still, addition of the protein environment had effects on the active-site geometry. The reason this does not affect the total barrier height is that when comparing transition state and reactant, the protein effect appears to be relatively constant. 

Theoretical calculations have been carried out on a number of zinc-containing enzymatic systems. For example, calculations on the mechanism of the Cu/Zn enzyme show the importance of the full protein environment to get an accurate description of the copper redox process, i.e., including the electronic effects of the zinc ion.989 Transition structures at the active site of carbonic anhydrase have been the subject of ab initio calculations, in particular [ZnOHC02]+, [ZnHC03H20]+, and [Zn(NH3)3HC03]+.990... 

Having an increased or elevated reactivity. This term has been used in reference to the relative activity of amino acyl residues at the active sites of enzyme. The immediate environment (Le., the microenvironment) may allow simple reagents to react faster with the amino acid than would normally be expected. Thus, in labeling of proteins with active site-directed reagents, an investigator should always consider the basis of increased reactivity Is it due to facilitation of the reaction by increased affinity (Le., affinity labeling), or is it due to increased activity of the amino acyl side chain (e.g., perhaps increased nucleophilicity due to the microenvironment). 

One of the questions surrounding the mechanism of tyrosinase concerns the initial site of attack. As a control, LFMD simulations of a model for the sTy active site, Meim6 (Fig. 28), give identical behavior for each Cu center consistent with its symmetry. In contrast, the LFMD simulations clearly distinguish the two copper sites in the sTy enzyme which must result from the protein environment (Fig. 29). 

Locating minima is not always straightforward since a reaction surface is usually complex, and a geometry optimization calculation will only locate minima close to the starting point. It is usually not feasible to systematically explore all possible conformers, so chemical intuition and corroborative evidence from experiment play important roles. A nice example is the identification of the coordination geometry of oxo-iron(IV) intermediate in TauD (22). As mentioned above, during optimization of enzyme active sites, key atoms are sometimes fixed to mimic the constraints that the protein environment exerts on the active site (20). 

It was noted that the conformation of an oligosaccharide may change depending on the protein environment where it is bound [162], The changes in conformation may cause enzymes to exhibit different activities depending on the site where the oligosaccharide is bound. This was called site directed processing by enzymes. 

Galactose oxidase hinds a single copper ion within Domain 11 on the axis of the wheel. The active site (Fig. 5) is unhke any other biological copper complex, an appropriate distinction for this remarkable enzyme. To explore the site in more detail, the protein environment of the mononuclear copper center may be separated into (A) direcdy coordinated metal hgands (hrst shell, inner sphere interactions) and (B) the extended active site environment (the second shell or outer coordination sphere). 

As discussed in the following sections, the catalytic diversity of enzymes containing Type 2 copper is striking. Much progress has been made in understanding how the protein environments modulate a tetragonal Cu site to achieve these varied activities. Key information has been obtained from NMR and/or X-ray structures of the Type 2 enzymes. 

To the best of our knowledge, no consensus has been reached in the hterature on methanol electro-oxidation by MDH enzymes. Hence, a detailed theoretical investigation is carried on the already proposed methanol oxidation mechanisms (A-E and H-T) by using more extended MDH active site models considering protein environment. 

The chemical modification of redox enzymes with electron relay groups permits the mediated electron transfer and the electrical wiring of the proteins [83-85] (Figure 5A). The covalent attachment of electron-relay units at the protein periphery, as well as inner sites, yields short inter-relay electron-transfer distances. Electron hopping or tunneling between the periphery and the active site allows electrical communication between the redox enzyme and its environment. The simplest systems of this kind involve electron relay-functionalized enzymes diffusionally communicating with electrodes [83], but more complex assemblies including immobilized enzymes have also been reported. 

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