Click image for a view of a Zinc metalloenzyme (CPA) active site
Metals play roles in approximately one-third of the known enzymes. Metals may be a co-factor or they may be incorporated into the molecule, and these are known as metalloenzymes. Amino Acids in peptide linkage posses groups that can form coordinate-covalent bonds with the metal atom. The free amino and carboxy group bind to the metal affecting the enzymes structure resulting in its active conformation (2).
Metals main function is to serve in electron transfer. Many enzymes can serve as electrophiles and some can serve as nucleophilic groups. This versatility explains metals frequent occurrence in enzymes. Some metalloenzymes include hemoglobins, cytochromes, phosphotransferases, alcohol dehydrogenase, arginase, ferredoxin, and cytochrome oxidase.
Carboxypeptidase A is a zinc metalloenzyme that breaks peptide linkages in the digestion of proteins. The zinc ion that the enzyme contains in its active site plays a key role in that function.
Metalloenzymes can be regulated in several ways since they are such a diverse group. One way metalloenzymes are regulated is the pH level. The pH level can disrupt the electron flow that the metal would normally help facilitate. In this way the pH level could inhibit the overall effectiveness of the metalloenzyme.
Transition state analogs play a key role in the competitive inhibition of metalloenzymes because they mimic the structure of the substrates transition state in the reaction of enzyme and substrate.
Metalloenzymes such as the ones containing zinc can also be regulated by diet. The source of zinc in humans is almost entirely through diet. Without proper intake of metals such as zinc in a persons diet, the activity of the enzyme would be inhibited.
One thing to keep in mind while studying metalloenzymes is that they are incredibly diverse and function in a multitude of important physiological processes
Click image for a view of a Zinc metalloenzyme (CPA) active site
Metalloenzymes are proteins which function as an enzyme and contain metals that are tightly bound and always isolated with the protein. In proteins such as hemoglobins and cytochromes, the metal is Fe2+ or Fe3+, and it is part of the heme prosthetic group. In other metalloenzymes the metal is built into the structure of the enzyme molecule. The metal ion can not be removed with out destroying the structure of the enzyme. Metals built into the molecule include: most phosphotransferases, containing Mg2+; alcohol dehydrogenase, Zn2+; arginase, Mn2+; ferredoxin, Fe2+; and cytochrome oxidase, Cu2+ (9).
Metals are usually found in the active site of the enzyme. The metals resemble protons (H+) in that they are electrophiles that are able to accept an electron pair to form a chemical bond. In this aspect, metals may act as general acids to react with anionic and neutral ligands (2).
Metal's larger size relative to protons is compensated for by their ability to react with more than one ligand. Metals typically react with two, four, or six ligands. A ligand is whatever molecule the metal interacts with. If a metal is bound with two ligands it will form a linear complex. If the metal reacts with four ligands the metal will be set in the center of a square that is planer or it will form a tetrahedral structure, and when six ligands react, the metal sits in the center of an octahedron.
By clicking the following image one can view a planar arrangement of and iron-porphyrin system:
Amino acids in their peptide linkage in proteins possess groups with the ability to bind to the metal resulting in coordinate-covalent bonds. The free amino and carboxyl groups in a protein can bind to the metal and this may bind the protein to a specific, active conformation (3). The fact that metals bind to several ligands is important in that metals play a role in bringing remote parts of the amino acid sequence together and help establish an active conformation of the enzyme.
Zinc is the metal incorporated in carboxypeptidase A. The zinc atom serves as a metal ion catalyst and promotes hydrolysis. The substrate fits into the hydrophobic pocket in carboxypeptidase A and zinc binds to the carboxyl group of the substrate to help stabilize the enzyme-substrate complex. In this example the zinc ion acts a generalized acid and stabilizes the developing O- as water attacks the carbonyl.
Zinc can also perform a different role in enzymes like the role it performs in carbonic anhydrase. Here the metal binds H2O and makes it acidic enough to lose a proton and form a Zn-OH group. The zinc metal serves as a nucleophile to the substrate. Since zinc has the ability to act as an electrophile or as the source of a nucleophilic group it is incorporated and used by many enzymes (10).
A four-subunit molecule, containing a iron atom in each subunit, in which each subunit binds a single molecule of oxygen. Hemoglobin transports oxygen from the lungs to the capillaries of the tissue.
Cytochromes are integral membrane proteins. Cytochromes contain iron which serves to carry electrons between two segments of the electron-transport chain. The iron is reversibly oxidizable and serves as the actual electron acceptor for the cytochrome.
The Mg2+ atom serves again in electron transfer.
A zinc metalloenzyme with broad specificity. They oxidize a range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones using NAD+ as a coenzyme.
The metal atom of Mn2+ is used in electron transfer.
An electron transferring proteins involved in one-electron transfer processes.
The copper ions easily accommodate electron removed from a substrate and can just as easily transfer them to a molecule of oxygen (10).
Carboxypeptidase A (CPA) is a zinc metalloenzyme that undergoes a large conformational change upon binding of the substrate that serves the purpose of bringing together the components of the active site. It is important to see that the zinc metal ion plays a key role in the catalytic process (10). Carboxypeptidase A is an exopeptidase which hydrolyzes the oligopeptides one at a time from the C-terminal end of the polypeptide chain. CPA is specific for large hydrophobic side chains while its closely related complimentary digestive enzyme, Carboxypeptidase B (CPB), is specific to basic residues. This complementary relationship between CPA and CPB is very similar to that of the closely related group of non-metalloenzymes of the digestive system, chymotrypsin and trypsin. However, chymotrypsin and trypsin are endopeptidases that catalyze the hydrolysis of non-terminal peptide bonds (3). As was stated CPA preferentially hydrolyzes peptides when the terminal residue is hydrophobic, either aromatic or branched aliphatic groups make favorable substituents. The binding is also stereospecific, as the side group must be in the L-configuration.
A good deal of movement takes place in CPA when it becomes bound to the substrate (figure 1). As the aromatic C-terminal side group of the substrate fits into a pocket in the interior of the enzyme molecule the Arg 145 in the active site a CPA mores 2 Å closer to interact with the substrates terminal carboxyl group. Tyr 248 swings down to place its hydroxyl near the nitrogen of the bond to be split, and the neighboring carboxyl oxygen becomes complexed with the Zn2+. As seen in figure 1 below.
The overall effect of the binding of substrate to CPA is the conversion of the enzyme cavity from a water-filled region to a hydrophobic region This change is made possible by the aforementioned ability to react with more than one ligand. In general, metal ions react with two, four, or six ligands. With this possibility in mind it shows us the importance of metals in there respective enzymes since they can both aid in binding and holding of the substrate in the active site but also aid in maintaining the tertiary structure of the enzyme itself (10).
Schematic views of the bound substrate, derived from structural studies, suggest that the Zn2+ plays a key role as an electrophilec catalyst. This view has also been supported by chemical studies of the enzyme-substrate complex. In a mechanism proposed by R. Breslow and D.L. Wernick, the peptide substrate binds so as to displace H2O form the Zn2+. This makes the carbon of the carbonyl more attractive for nucleophilic attack as it results in a greater partial positive charge on this carbon. Then a water molecule is delivered by the glutamate carboxylate (Glu 270). After delivery of the hydroxyl to the carbonyl and pickup of the first proton by Glu 270, there must be an additional proton transfer to permit cleavage, so both protons of H2O eventually are released. In the mechanism shown in Figure 2 this second proton is mediated through the hydroxyl group of Tyr 248, which functions as a general acid catalyst.
Approximately one-third of the known enzymes have metals as part of their structure, require that metals be added for activity, or are further activated by metals. In enzymes where a metal has been built into the structure of the enzyme molecule, the metal cannot be removed without destroying that structure. Such enzymes include the metalloflavoproteins, the cytochromes, and the ferredoxins. In enzymes where metals are required to be added for activity the metals react reversibly with proteins to form metal-protein complexes that constitute the active catalyst. In many instances, the complex represents a specific, catalytically active conformation of the protein; the role of the metal appears to be one of stabilizing that conformation (2).
Because the grouping, metalloenzymes, is so large and broad it would be almost impossible to explain how all of them can be controlled and regulated. In light of this, it is important to instead mention how the important functions of metals in enzymes can be disrupted and thus inhibited. Metals resemble protons (H+) in that they are electrophiles that are capable of accepting an electron pair to form a chemical bond. In doing so, metals may act as general acids to react with anionic and neutral ligands. This characteristic of metals is helpful in enzymatic structure and function but makes the enzyme it is part of pH dependent. Changes in pH can disrupt this electron flow that the metal would normally help facilitate and thus inhibit the overall effectiveness of the metalloenzyme.
Also, because of the variability inherent to the metal's ability to react with more than one ligand, you see metals as part of the active site in many metalloenzymes. Competitive inhibitors in the form of transition-state analogs are compounds believed to look like the substrate in its transition state. To be effective the transition-state analog must not be susceptible to reaction by the enzyme. Competitive inhibition, via transition-state analog, has been exhibited in the reaction of Carboxypeptidase A by a phosphorus molecule constructed by Paul Bartlett, which can be seen in Figure 3 (10).
It functions will in inhibition of CPA because the phosphorus atom, with its attached oxygens and nitrogen, resembles the tetrahedral carbon atoms in the two intermediates of Figure 2, and the transition states of all three steps (10).
As aforementioned, metals play a large role in the activity of a multitude of biological molecules. The source of zinc in humans is almost entirely through diet and without the intake of metals such as zinc in the diet one almost certainly inhibits the production and/or activity of many vital enzymes. Among enzymes that would not be produced by the body were it not for the presence of zinc in the body are carbonic anhydrase, the carboxypeptidases, alkaline phosphatase, lactic acid, and alcohol dehydrogenases.
The recommended daily dietary allowance for zinc is 15 mg., with 20 and 25 mg. During pregnancy and lactation. The average adult human being ingests 12 to 20 mg. Of zinc per day. Deficiencies in dietary zinc intake can result in stunted growth, enlarged liver and spleen, and underdevelopment of genitals and secondary sex characteristics. Outside of dietary intake deficiencies in zinc, and thusly in enzymes that contain zinc, can be caused by the excretion of zinc in perspiration, or by blood loss if there is parasite infection.
There is also increasing evidence that zinc plays an important role in protein biosynthesis and utilization. The addition of small amounts of zinc to a diet containing suboptimal amounts of a vegetable protein, as indicated by the growth of young rats, causes a pronounced increase in protein utilization and growth. This defect may result from a failure in adequate RNA synthesis. Zinc apparently inhibits the enzyme ribonuclease. Thus, in zinc deficiency, excessive destruction of RNA could occur. This demonstrates that the dietary intake of metal is not only important for the production of key enzymes but also for the inhibition of many others.
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5. http://expasy.hcuge.ch:80/pub/Graphics/IMAGES/GIF/---site for Carboxypeptidase-A--- 1.gif
6. http://www.scripps.edu/dbg/activesi.jpg
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