Catalytic centers enzymes

When induced in macrophages, iNOS produces large amounts of NO which represents a major cytotoxic principle of those cells. Due to its affinity to protein-bound iron, NO can inhibit a number of key enzymes that contain iron in their catalytic centers. These include ribonucleotide reductase (rate-limiting in DNA replication), iron-sulfur cluster-dependent enzymes (complex I and II) involved in mitochondrial electron transport and cis-aconitase in the citric acid cycle. In addition, higher concentrations of NO,... 

Monooxygenases (MOs) Enzyme systems of the endoplasmic reticulum of many cell types, which can catalyze the oxidation of a great diversity of lipophilic xenobiotics, are particularly well developed in hepatocytes. Forms of cytochrome P450 constitute the catalytic centers of monooxygenases. 

True examples of single-site catalysts are enzymes, where active sites are made mainly by metalhc centers (mono- or polynuclear species) whose coordination sphere is completely defined by ligands [1-4]. The strength of enzymes is the combined effect of metal center activity with the specific behavior of metal coordination sphere hgands. These species play a key role, being optimized to create an environment suitable for (i) metal centers approaching and coordinating by reactants (ii) product removal from the catalytic centers at the end of the reaction in order to avoid further reactions. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

The catalytic center of the ribosome, where the protein is actually made, is now seen to consist of RNA, not of a protein enzyme The many... 

LOXs are proteins containing a single atom of nonheme iron in catalytic center, with the ferric enzyme in an active form. The free radical-mediated mechanism of LOX-catalyzed process may be presented as follows (see also Figure 26.1) ... 

The interaction within the active site can be either in the form of covalent binding or in the form of quasi-irreversible (tight but slowly reversible) binding, and it can involve the protein residues, the porphyrin moiety or the catalytic center (heme iron) [8]. CYP inactivation follows a stoichiometry of one substrate molecule per enzyme molecule inactivated. To measure the stoichiometry of the inactivation, it is necessary to trap all molecules that are not specifically bound to the active site, by using an appropriate scavenger, normally GSH. 

The conformational fixation and spatial organization of the catalytic group are important features of the enzyme active site. However, they are not realized by the use of conventional surfactant micelles. Synthetic bilayer membranes are better organized than surfactant micelles. Thus highly organized catalytic centers may be prepared in the future from synthetic bilayer systems. 

In other words, we try to mimic enzymes by attaching centers for homogeneous catalysis to polymer chains we want to learn from nature how to conduct chemical processes in a cleaner, more selective and milder way. In this respect it is of great importance that we can adapt, just like in enzymes, the micro-environment of the catalytic centers by modification of neighbouring polymer chain segments. 

All enzymatic processes are complex reactions that involve more than one step. The substrate first binds to the enzyme, in the second step reaction occurs, and finally products are released from the enzyme. This all happens at a catalytic center in the enzyme which is termed the active site. Enzymes are usually very large molecular systems, and may contain anywhere between several and several hundred aminoacids. The active site is usually buried inside a bulky three dimensional structure that shields the reactant-active site complex from the surrounding bulk phase aqueous solution. It typically contains several aminoacids that are vital for... 

Fig. 3.13. The 3D structure of neprilysin (enkephalinase, EC 3.4.24.11) obtained from ldmt.pdb [79]. This structure was determined for the enkephal-inase-phosphoramidon complex, but the inhibitor has been removed to unmask the catalytic center, a) Structure of the enzyme, with the Glu and the two His residues of the HEXXH zinc-binding motif shown in blue, b) Zoom on the catalytic center, revealing the spatial arrangement of the zinc-binding residues.

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

As stated earlier, to make such a catalytic center is not a simple job. However, it can be said with confidence that, in view of the successes with some of the structures described here, the goal appears a great deal more attainable now than it was two decades ago when Morawetz, Bender, Bruice, and others began looking at enzyme models. 

M. Fabian and co-workers have studied the protein s role in internal electron transfer to the catalytic center of cytochrome c oxidase using stopped-flow kinetics. Mitochondrial cytochrome c oxidase, CcO, an enzyme that catalyzes the oxidation of ferrocytochrome c by dioxygen, is discussed more fully in Section 7.8. In the overall process, O2 is reduced to water, requiring the addition of four electrons and four protons to the enzyme s catalytic center. Electrons enter CcO from the cytosolic side, while protons enter from the matrix side of the inner mitochondrial membrane. This redox reaction. 

Using an approach that has been successful with protein enzymes and other ribozymes, researchers have used hammerhead X-ray crystallographic structures to identify sites around the catalytic center where mutation might yield... 

There are several demonstrations that cytochrome cdi catalyzes the reduction of molecular oxygen to water. Exactly how the enzyme catalyzes this reaction is of some interest, because the crystal structure shows that the catalytic center is mononuclear and expected to handle one electron at a time. If we assume that electron transfer between subimits cannot occur, then only two of the four electrons required for reduction of one oxygen atom can obviously be stored on one subimit of the enz5une before reduction of oxygen commences. Thus, it might be anticipated that some intermediates of oxygen reduction are relatively long-lived. 

A now classical example of the power of equilibrium dialysis is the determination by Englund et al that DNA polymerase binds all four nucleoside 5 -triphosphate substrates at the same subsite in the enzyme s catalytic center. See also Womack-Colowick Technique... 

The specific function of the SH3 domain is based on increased substrate specificity of tyrosine kinases in this case (Shokat, 1995). In classical enzymes, the substrate binding site and the catalytic center are close together and the substrate binding site is generally highly specific for a particular substrate. The situation is different for tyrosine kinases. Here, the substrate binding site near the catalytic center shows moderate selectivity. The specificity of the reaction is increased, however, by mediation of asso-... 

It is now clear that in addition to their widespread involvement in electron transfer pathways, iron-sulfur clusters function as catalytic centers in a wide variety of enzymes. The first example of such an enzyme is aconitase. It was at first thought that the role of the iron-sulfur group was regulatory, but it is now clear that in this enzyme the iron-sulfur group is part of the catalytic site. One of the iron atoms can coordinate water or hydroxyl and plays a key role in the isomerization catalyzed by the enzyme (Emptage et al., 1983). 

The function of zinc ions may be either catalytic or stractural. Enzymes with a co-catalytic center of two or even three zinc ions in close proximity are also known. In a new type of zinc-binding site, the protein interface, zinc ions are fixed at the interface of two proteins with the aid of amino acid residues. The ligand residues are usually His, Asp, Glu or Cys, which interact via nitrogen, oxygen or sulfur donors with the metal ion. In catalytic binding sites. His coordination dominates and an additional reactive water molecule is bound. 

---