Enzyme active center

Phosphoglycomutase (bacterial) One Meth residue in ezyme active center Enzyme inactivitation, abolition of glucose glycolytic metabolism (S59)... 

Despite the large size of an enzyme molecule, there is reason to believe that there are only one or a few spots on its surface at which reaction can occur. These are usually referred to as active centers. The evidence for this view of enzyme reactions comes from many kinds of observations. One of these is that we can often stop or slow down enzyme reactions by adding only a small amount of a false substrate. A false substrate is a molecule that is so similar to the real substrate that it can attach itself to the active center, but sufficiently different that no reaction and consequently no release occurs. Thus, the active center is blocked by the false substrate. 

It seems reasonable that an enzyme which used poraaminobenzoic acid as a substrate might be deceived by sulfanilamide. The two compounds are very similar in size and shape and in many chemical properties. To explain the success of sulfanilamide, it is proposed that the amide can form an enzyme-substrate complex that uses up the active centers normally occupied by the natural substrate. 

Usually fairly high concentrations of such a drug are needed for effective control of an infection because the inhibitor (the false substrate) should occupy as many active centers as possible, and also because the natural substrate will probably have a greater affinity for the enzyme. Thus the equilibrium must be influenced and, by using a high concentration of the false substrate, the false substrate-enzyme complex can be made to predominate. The bacteria, deprived of a normal metabolic process, cannot grow and multiply. Now the body s defense mechanisms can take over and destroy them. 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

The [NiFe] hydrogenase from D. gigas has been used as a prototype of the [NiFe] hydrogenases. The enzyme is a heterodimer (62 and 26 kDa subunits) and contains four redox active centers one nickel site, one [3Fe-4S], and two [4Fe-4S] clusters, as proven by electron paramagnetic resonance (EPR) and Mosshauer spectroscopic studies (174). The enzyme has been isolated with different isotopic enrichments [6 Ni (I = I), = Ni (I = 0), Fe (I = 0), and Fe (I = )] and studied after reaction with H and D. Isotopic substitutions are valuable tools for spectroscopic assignments and catalytic studies (165, 166, 175). 

The protein from D. desulfuricans has been characterized by Mbss-bauer and EPR spectroscopy 224). The enzyme has a molecular mass of approximately 150 kDa (three different subunits 88, 29, and 16 kDa) and contains three different types of redox-active centers four c-type hemes, nonheme iron arranged as two [4Fe-4S] centers, and a molybdopterin site (Mo-bound to two MGD). Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. 

FIGURE 10.3 Acetylcholinesterase structure of catalytic triad. The structure of the catalytic triad of the active center of the enzyme is shown (from Sussman et al. 1991). 

The inhibition of brain cholinesterase is a biomarker assay for organophosphorous (OP) and carbamate insecticides (Chapter 10, Section 10.2.4). OPs inhibit the enzyme by forming covalent bonds with a serine residue at the active center. Inhibition is, at best, slowly reversible. The degree of toxic effect depends upon the extent of cholinesterase inhibition caused by one or more OP and/or carbamate insecticides. In the case of OPs administered to vertebrates, a typical scenario is as follows sublethal symptoms begin to appear at 40-50% inhibition of cholinesterase, lethal toxicity above 70% inhibition. 

Among several applications, Fe-based hydrogenases play a central role in the stepwise reduction of CO2 to methane. This process is accomplished through various types of Fe-hydrogenases however, in most of these enzymes, the active center is either a binuclear Fe-Fe- or an Ni-Fe-complex. Although the exact... 

After the nucleophilic attack by the hydroxyl function of the active serine on the carbonyl group of the lactone, the formation of the acyl-enzyme unmasks a reactive hydroxybenzyl derivative and then the corresponding QM. The cyclic structure of the inhibitor prevents the QM from rapidly diffusing out of the active center. Substitution of a second nucleophile leads to an irreversible inhibition. The second nucleophile was shown to be a histidine residue in a-chymotrypsin28 and in urokinase.39 Thus, the action of a functionalized dihydrocoumarin results in the cross-linking of two of the most important residues of the protease catalytic triad. 

Apart from the isomeric relation between 7 and 1, the appearence of the ternary associate now showing coordinatoclathrate properties gives a reasonable motive for putting up these compounds for discussion here. The dimer formation of carboxylic acids known from the related inclusions of 1 and 26 does not occur here. Instead, one observes a well-balanced system of H-bonds between groups of different acid/base properties. It is left to future studies to find other acid/base combinations which give a comparable situation. Actually, such H-bonded systems remind one of the multiple non-bonded interactions at the active centers of enzymes. 

A number of molybdenum-containing hydroxylases catalyzing the first hydrox-ylation step of N-containing compounds have been characterized thoroughly (e.g., carbazole [314], quinoline [327], and indole [350]). The enzyme s redox-active has been described as a molybdenum ion site coordinated to a distinct pyranopterin cofactor (two different [2Fe2S] centers) and in most cases, flavin adenine dinucleotide centers. This active center transfers electrons from the N-heterocyclic substrate to an electron acceptor, which for many molybdenum hydroxylases is still unknown [350],... 

In the first family, the metal is coordinated by one molecule of the pterin cofactor, while in the second, it is coordinated to two pterin molecules (both in the guanine dinucleotide form, with the two dinucleotides extending from the active site in opposite directions). Some enzymes also contain FejSj clusters (one or more), which do not seem to be directly linked to the Mo centers. The molybdenum hydroxylases invariably possess redox-active sites in addition to the molybdenum center and are found with two basic types of polypeptide architecture. The enzymes metabolizing quinoline-related compounds, and derivatives of nicotinic acid form a separate groups, in which each of the redox active centers are found in separate subunits. Those enzymes possessing flavin subunits are organized as a2jS2A2, with a pair of 2Fe-2S centers in the (3 subunit, the flavin in the (3 subunit, and the molybdenum in the y subunit. 

Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear et al. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci USA 1988 85 9386-9390. 

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. 

Cross-linking reactions are carried out under relatively severe conditions. Nevertheless, it must be taken into account that such harsh conditions may alter the conformation of the active center of the enzyme leading to a significant loss of activity. 

One of the best ways to ensure retention of activity in protein molecules is to avoid doing chemistry at the active center. The active center is that portion of the protein where ligand, antigen, or substrate binding occurs. In simpler terms, the active center (or active site) is that part that has specific interaction with another substance (Means and Feeney, 1971). For the preparation of enzyme derivatives, it is important to protect the site of catalysis where conversion of substrate to product happens. For instance, when working with antibody molecules, it is crucial to stay away from the two antigen binding sites. 

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