Enzyme/enzymatic metalloenzyme

Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

In zinc metalloenzymes. zinc is a selective stoichiometric constituent and is essential for catalytic activity. It is frequently present in numerical correspondence with the number of active enzymatic sites, coenzyme binding sites, or enzyme subunits Removal of zinc results in loss of activity. Inhibition by metal complexing agents is a characteristic feature of zinc metalloenzymes. However, no direct relationship holds between the inhibitory effectiveness of these agents and their affinity for ionic zinc. Although zinc is the only constituent of zinc metalloenzymes in vivo, it can be replaced by other metals m vitro, such as cobalt, nickel, iron, manganese, cadmium, mercury, and lead, as m the case of carboxy-peprida.ses. 

Manganese The human body is estimated to contain a total of 10-20 mg Mn. The RDA is between 2 and 5 mg per day. Manganese is involved in many enzymatic processes as a metalloenzyme or as a cofactor. In contrast to the Mn-activated enzymes, there are few Mn metalloenzymes (arginase, pyruvate carboxilase, and Mn-superoxide dismutase) [14]. The content of Mn in milk responds rapidly to changes in dietary intakes of this metal. 

N-substituted iron porphyrins form upon treatment of heme enzymes with many xenobiotics. The formation of these modified hemes is directly related to the mechanism of their enzymatic reactivity. N-alkyl porphyrins may be formed from organometallic iron porphyrin complexes, PFe-R (a-alkyl, o-aryl) or PFe = CR2 (carbene). They are also formed via a branching in the reaction path used in the epoxidation of alkenes. Biomimetic N-alkyl porphyrins are competent catalysts for the epoxidation of olefins, and it has been shown that iron N-alkylporphyrins can form highly oxidized species such as an iron(IV) ferryl, (N-R P)Fe v=0, and porphyrin ir-radicals at the iron(III) or iron(IV) level of metal oxidation. The N-alkylation reaction has been used as a low resolution probe of heme protein active site structure. Modified porphyrins may be used as synthetic catalysts and as models for nonheme and noniron metalloenzymes. 

Metalloenzymes or metallocoenzymes are involved in a great deal of enzymatic activity, which depends on the presence of metal ions at the active site of the enzyme or in a key coenzyme. Of the latter, the best known is vitamin B12, which contains cobalt. Important metalloenzymes include carboxypeptidase (Zn), alcohol dehydrogenase (Zn), superoxide dismutase (Cu, Zn), urease (Ni), and cytochrome P-450 (Fe). 

Finally in this section, we note the remarkable prevalence of tetrahedral four-coordination in zinc(II) chemistry see Zinc Inorganic Coordination Chemistry), to an extent comparable with cobalt(II), although d enjoys no advantage from CFSE. Tetrahedrally bonded zinc(II) is found in many metalloenzymes see Zinc Enzymes) and can often be replaced by cobalt without loss of enzymatic activity. This can be attributed to d-p mixing. In tetrahedral symmetry Td), the nAxy,xz,yz and(n + l)Px,y,z orbitals each constitute a threefold-degenerate t2 set and become mixed. The LF t2 orbitals are thus lowered in energy, and the d configuration (where t2 is filled) is consequently stabilized. 

Biochemistry. Zinc is essential for many biological functions in man and animals. Major functions of zinc in human and animal metabolism appear to be enzymatic. There are now more than 70 metalloenzymes known to require zinc for their functions (12,13). Zinc enzymes are known to participate in a variety of metabolic processes including carbohydrate, lipid, protein, and nucleic acid synthesis or degradation. The metal is present in several dehydrogenases, aldolases, peptidases, and phosphatases. 

The assessment of functional significance of associations between metals and proteins is greatly facilitated when the pure proteins under study exhibit specific enzymatic function. These enzymes may fall in either group of proteins described above. Highly purified proteins which contain a metal firmly and apparently uniquely bound—metalloenzymes—represent particularly useful model systems for the study both of interactions of proteins with metals and of the biological effects of metals. Since the members of this group are structural and functional units, at least three primary parameters can be measured independently to ascertain the interdependence between structure and function ) The protein, 2) the metal, and (3) the activity. 

Metal-enzyme complexes, a subgroup of metal-protein complexes, exhibit enzymatic activity consequent to readily dissociable combination with a variety of metal ions. Many of these studies have been performed with unpurified enzymes, and, even when pure enzymes were used, the stoichiometry of the interaction of the metal and enzyme has not been measured. Enhancement of enzymatic activity as a result of the addition of metal ions and its partial loss on their removal has been the chief criterion of assessment of physiological significance. Only in a few instances, e.g., enolase, has the stability and stoichiometry been studied in relation to function (Malmstrom, 1953, 1954). The study of metal complexes and particularly metal chelates (Bjerrum, 1941 Martell and Calvin, 1952 Calvin, 1954) has provided both new experimental and new theoretical backgrounds for the study of metals in relation to the specificity of enzyme action, metal-enzyme (Calvin, 1954), metal-substrate (Najjar, 1951), and metalloenzyme interaction, as well as metal-enzyme inhibition (James, 1953). 

The kinetics and electronic mechanisms of conventional chemical catalysts- are contrasted with those in enzymes. The analogy between certain attributes of surfactants and phase-transfer catalysis and enzyme active sites are made and the limitations of surface catalysts and zeolites are pointed out. The principle features that give enz3nnes their unusual rate enhancements and remarkable specificity are discussed and ways in which these can be realized in man-made catalysts are proposed. The catalytic activation of CO2 by both enzymatic and non-enzymatic means, including a detailed analysis of the electronic reaction sequence for the metalloenzyme carbonic anhydrase, is used to illustrate the above themes. 

The additional comment that the high aflSnity of metalloenzymes for their metals as "compared with the stability of chelates which use the same ligands, argues against a thermodynamically strained coordination is similarly not relevant and based upon a misinterpretation of the entatic site hypothesis. Entasis implies that the difference in energy between the ground state and transition state for the enzymatic reaction is reduced, not that the metal-enzyme complex is thermodynamically less stable, as was inferred. Indeed, there is no reason to suppose that the distorted environment of a metal ion in an enzyme as opposed to a simple metal complex leads necessarily to an increase in free energy. The studies of alkaline phosphatase just presented certainly seem consistent with the entatic state hypothesis. 

Carbonic anhydrase (CA) is a zinc metalloenzyme involved in mammalian respiration, which catalyzes the hydration of carbon dioxide. Copper-complexed TPPC, competitively inhibits CA enzymatic activity as does copper-complexed TPPSj [32]. Experiments comparing the spectrophotometric characteristics of the two porphyrins in the presence of CA and apo-CA indicate that the zinc atoms in the active site of the enzyme are indeed involved in the interaction between the porphyrins and the enzyme. The metal-free porphyrins TPPSj and TPPC, do not inhibit the enzymatic activity of CA. Further, the spectrophotometric characteristics of these porphyrins in the presence of apo-CA were identical to those in the presence of wild-type CA, indicating the lack of involvement of the active site-coordinated zinc in the porphyrin-enzyme interaction for metal-free porphyrins. 

It has been the intention of the author in this review to examine the roles played by zinc ion in homogeneous solution catalysis both for small molecule-zinc ion complexes and for zinc-metalloenzymes. Emphasis is placed on the integration of physical-inorganic mechanistic concepts derived from studies on small molecule systems with the accumulated kinetic, chemical, and structural information available on select enzyme examples in order that reasonable mechanistic hypotheses might be developed for the roles played by zinc ion in enzymatic catalysis. 

OPH catalyzes the hydrolysis of organophosphonates with P-0, P-F, P-S, and P-CN phosphoryl bonds such as those of coumaphos, sarin, VX, and paraoxon. Hydrolysis is facilitated by two metal atoms in the active site and results in the release of two protons (79). In the native dimeric form of the enzyme, zinc is coordinated to histidine residues in the active site. Reconstitution of the apo-entyme with cobalt or cadmium results in higher enzymatic activity than that observed in the zinc-metalloenzyme (20). 

The study of biomimetics can be of great benefit for the understanding of enzymatic reactions. The term biomimetic refers, in the context of this work, to a compound that mimics structural, functional and spectroscopic properties of an enzyme [67]. Often only one or two of these aspects are achieved for a model system and they usually display substantially lower activity. There are, however, advantages over the enzyme model complexes are generally more stable and robust than their enzymatic counterpart, they can be readily crystalUzed and provide easy accessible structural information on metal ion coordination. Also as these model systems are considerably less complex, kinetic and spectroscopic data interpretation is simplified and— by comparison to data derived for the enzyme— the mechanism of action and structural features can be elucidated and thus related back to the parent metalloenzyme. Also models can be obtained on a larger scale and are often less costly to synthesize, a distinct benefit for potential applications. A few structures of model complexes for dinuclear hydrolytic enzymes are shown in Fig. 1.4. The approaches for ligand and complex design are diverse. 

Trace metals including Mg, Zn, Cu, Mn, Fe, and Co are employed as cofactors in a number of enzymatic reactions. Indeed, for many enzymes the only cofactor is a metal ion. Two broad classes of metalutilizing enzymes are distinguishable by experimental means. These are the metalloenzymes where the metal is bound to the apoenzyme with sufficient strength to survive purification procedures and is present in a definite proportion to the protein, and the metal enzyme complexes where the metal, although easily dissociable, is required for full expression of enzyme activity. 

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