Pet Enzyme lesson plan Introduction A. Enzymes as catalysts 1. Reactions may be thermodynamically favorable but not spontaneous 2. Kinetics/ and Ea of activation 3. Activation energy plot 4. Enzymes lower activation energy 5. Enzymes are specific to a given substrate 6. Catalyzed by a. Deforming substrates b. Creating a favorable chemical environment 7. Regulation 8. Cofactors coenzymes 9. Inhibition a. Irreversible b. Competitive non competitive c. Allosteric inhibiton d. Allosteric activiation e. Feedback inhibition f. Cooperative inhibition Discussion: Carbonic Anhydrase catalyzes the removal of carbon dioxide from the body. Without this vital enzyme, carbon dioxide concentrations would quickly reach fatal levels in our bodies. Carbonic anhydrase also helps maintain the correct ph levels in the body as it can facilitate the movement of protons across membranes and throughout the body. Increasing or decreasing the local [H + ] affects many ph dependant reactions throughout our body. The enzyme was originally discovered in the epithelial cells of cows. In mammals it exists in the α form ( mammalian form), (the β form) is found in plants, and the γ form is found in bacteria (see Figure 1a). Although the 3 forms of the enzyme share few structural similarities, they all contain a zinc atom as a ligand with which they catalyze the removal of carbon dioxide. The form of mammalian carbonic anhydrase varies depending on its location in the body. Three variations of the enzyme (isozymes) are found in the cytosol, another five are membrane bound. There is an izozyme complexed in the mitochondria and one is secreted in saliva. Carbonic anhydrase has other vital functions in our body. Its ability to control ph and proton concentration means that it can affect many other bodily functions. For example, the water content in our kidneys and eyes depends on the ratio of protons to bicarbonate ions. Glaucoma (the buildup of pressure in the eyes as a result of increased fluid is controlled through the inhibition of carbonic anhydrase. Carbonic anhydrase isozyme in saliva helps maintain a neutral ph in the mouth. It helps maintain acidic conditions in the stomach and the alkalinity of pancreatic juices. The outline to this lesson should serve as a general review of enzymes. The focus is on carbonic anhydrase, its structure and function.
Answers to suggested questions: 1. Carbonic anhydrase catalyzes the removal of carbon dioxide from the bloodstream. With the aid of its zinc ligand, the enzyme reacts CO 2 with a hydroxyl ion that is bound to the Zn 2+ ion. The presence of the positively charged zinc ion it polarizes a bound water molecule thus making the proton very acidic. The ionization of the proton is facilitated by (Glu 106 or Glu 117) thus creating the Zn-OH -1 complex. The reaction of the Zn complex with a polarized carbon dioxide molecule creates the bicarbonate ion. The bicarbonate leaves and travels to the lungs where it react with H + protons (assisted by hemoglobin) to reform carbon dioxide and water (via carbonic acid formation). (See figure 1: Carbon Dioxide Pathway). The overall reaction is: [E-Zn-OH - ] + CO 2! HCO 3 - + [Zn-OH-H] Followed by [Zn-OH-H]! [Zn-OH - ] + H + 2. The primary structure consists of 260 residues. See Figure 2. 3. The secondary structure contains the following elements: 2 beta sheets: o Sheet A: 2 parallel strands o Sheet B: 13 mixed strands 4 beta hairpin turns 5 beta bulges 9 helices 4 helix-helix interacts 23 beta turns 3 gamma turns (Figure 3 has a diagram of the layout of these structures. Note that it starts at residue # 4.). The Figure 3: Diagram of Secondary Structure The figure above shows the details of the secondary structure of carbonic anhydrase.
The key for this figure is as follows: 9 Helices H 1 H 9 : Correspond to the 15 Beta Strands β correspond to the 23 Beta turns 4 hairpin turns ( long U shapes 3 γ Turns 4 Helix-Helix interactions: H 1 -H 2 ; H 6 -H 7 ; H 6 -H 9 and H 7 -H 9 Although figures 4 4c are illustrations of the tertiary structures, they are useful representations of the elements of the secondary structure. 4. Figure 5 is The Hydropathicity Plot. Hydropathicity refers to the hydrophilic, hydrophobic or amphiphilic of segments of the protein chain. 5. Figures 4, 4a, 6, 6a,6b are all representations of the tertiary structure of the enzyme. Figure 4a shows the location of the zinc ligand. In the middle of the figure one can see the 13 strands of the B sheet, figure 4b looks at the same area, from the reverse side. 6. Figures 4a, 4c; figures 6 to 6c and 7 to 7b show the location of the zinc ligand from different perspectives. Table below shows the connectivity and distance of the ligand to nearby residues. Histidines 94, 96 and 119 are directly complexed to the zinc ion. Threonine119 is indirectly connected to the ion. Mammalian α canhydrase has one ligand, the β has four and γ has three ligands ( see Figure 1a). 7. Figure 8 shows the detailed reaction mechanism. Figure 8a shows the Histidine Swing that removes the proton from the complex. 8. This enzyme is so efficient that it is referred to as catalytic perfect. The kinetic definition of this defined by considering the k cat to K m where k cat is the catalytic constant (number of reactions at each active site per unit of time) and K m is the substrate concentration at which the V max is half maximal. An enzyme with a very high k cat / K m ratio catalyzes a reaction almost evertime a it encounters a substrate molecule. Enzymes with k cat / K m ration in the 10 8 to 10 9 ( M -1 s -1 ) are characterized as catalytically perfect. Kinetic Data for Carbonic Anhydrase is below. Substrate K m (M) k cat (s -1 ) k cat / K m (M -1 s -1 ) CO 2 1.2 E 2 1.0 E 6 8.3 E 7-1 HCO 3 2.6 E -2 4.0 E 5 1.5 E 7 Voet & Voet,1990 p.337
9. Carbonic anhydrase can affect the ph, water content, and osmotic balance in the body. A deficiency or an overabundance of it can have profound affects on the health of an individual. There are therefore many medications that affect carbonic anhydrase activity. For instance: ACETAZOLAMIDE Acetazolam Dazamide Diamox StorzolamideDICHLORPHENAMIDE Daranide METHAZOLAMIDE MZM Neptazane are all anhydrase inhibitors. Regulation can help increase or decrease acidity and fluid imbalances. Acetazolamide for example is given as a treatment for glaucoma. http://www.nextmd.com/drugs/carbonic_anhydrase_inhibitors.asp Figure 9 has a complete listing of illnesses that are treated with anhydrase inhibitors:] There are four types of anionic compounds that work to inhibit carbonic anhydrase: mercuric ion Hg 2+, thiocyanate SCN ; AMS (3-acetoxymercuri- 4-aminobenzenesulfonamide) and Diamox (acetazolamide). These substances inhibit the enzyme by distorting the geometry of the Zn-OH environment (AMX, Diamox,SCN) or by altering the charge distribution at the Zn-OH site.
References: Web sites: http://www.rcsb.org/pdb/molecules/pdb49_1.html http://www.rcsb.org/pdb/molecules/pdb49_1.html http://www.ebi.ac.uk/interpro/potm/2004_1/page1.htm http://info.bio.cmu.edu/courses/03231/protstruc/bctmech.htm#bctmech. http://www.rcsb.org/pdb/molecules/pdb49_3.html 1CA2 at PDB: http://pdbbeta.rcsb.org/pdb/explore.do?structureid=1ca2 http://www.chem.uwec.edu/chem406/webpages/kat/inhibitors.html Textbooks Voet & Voet (1990): Biochemistry. John Wiley and Sons. New York. Campbell, R. 1990 Biology: fifth edition Addison Wesley Menlo Park CA. Garrett &Grisham: Principles of Biochemistry With a Human Focus1997) Brooks Cole Australia
Figure 1: Carbon Dioxide Pathway Figure 1a. Three Types of Carbonic Anhydrase α Carbonic Anhydrase β Carbonic Anhydrase γ Carbonic Anhydrase http://www.rcsb.org/pdb/molecules/pdb49_1.html
Figure 2: Primary Structure of Carbonic Anhydrase 260 residues 1 SHHWGYGKHN GPEHWHKDFP IAKGERQSPV DIDTHTAKYD PSLKPLSVSY SSTTT TTTTHHHHTG GGGSSS S EE TTTSEE TT EEEE 51 DQATSLRILN NGHAFNVEFD DSQDKAVLKG GPLDGTYRLI QFHFHWGSLD TT B EEEE SS EEEEE SSTTSEEEE TT S EEE EEEEEE SST 101 GQGSEHTVDK KKYAAELHLV HWNTKYGDFG KAVQQPDGLA VLGIFLKVGS T SEETT B SEEEEEE EEEGGGSSHH HHTTTTTSEE EEEEEEEEES 151 AKPGLQKVVD VLDSIKTKGK SADFTNFDPR GLLPESLDYW TYPGSLTTPP GGGHHHHH HTGGGTTBT EEE S GG GG S EE EEEE SSTT 201 LLECVTWIVL KEPISVSSEQ VLKFRKLNFN GEGEPEELMV DNWRPAQPLK Reference Table for One Letter Codes in Structure Amino acid Three letter code One letter code alanine ala A arginine arg R asparagine asn N aspartic acid asp D asparagine or aspartic acid asx B cysteine cys C glutamic acid glu E glutamine gln Q glutamine or glutamic acid glx Z glycine gly G histidine his H isoleucine ile I leucine leu L lysine lys K methionine met M phenylalanine phe F proline pro P serine ser S threonine thr T tryptophan try W tyrosine tyr Y valine val V
Figure 4: Sheets and Helices in Carbonic Anhydrase http://cathwww.biochem.ucl.ac.uk/latest/domains/1c/1ca200.html Figure 4a. Beta Sheets and location of Zn ligand http://www.rcsb.org/pdb/molecules/pdb49_3.html
Figure 4b. Beta Sheets, Alpha helices and β turns http://www.arches.uga.edu/~obrien21/bcmb8010/structure.html Figure 4c. Alpha Helices http://www.ebi.ac.uk/thorntonsrv/databases/cgibin/pdbsum/getpage.pl?pdbcode=1ca2
Figure 5 : Hydropathicithy Plot
Figure 6: Tertiary Structure Asymmetric molecule a 8 helices are visible in this figure along with 7 beta sheets. The ligand is the grey atom. http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics;pdbid=1ca2;page=;pid=2651011251047 17&opt=show&size=250&cyl=1 Figure 6a. Asymmetric view 8 beta sheets, 5 helices: ligand is grey atom http://www.rcsb.org/pdb/cgi/explore.cg i?job=graphics;pdbid=1ca2;page=;pid =265101125104717&bio=1&opt=show &size=500
Figure 6c. Biological unit Zn ligand is in grey. http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics;pdbid=1ca2;page=;pid=2651011251047 17&bio=1&opt=show&size=500
Figure 7:Ligand Enzyme Complex: http://www.umich.edu/~caflab/images/caactivesite.jpg Figure 7a. Figure 7b. http://www.umich.edu/~caflab/images/ca.bmp
Figure 8: Reaction Mechanism. The are waters bound to the His 64 are ever present in the complex. They will form the water bridge, that will accept the proton product in step 5 of this reaction sequence. The hydrogen bonding that occurs at the nitrogen of His 64 polarizes water molecules. CO 2 Binding. The main chain NH of Threonine 199 orients and polarizes the CO 2 molecule. Hydrogen bonding between the NH of and the carbon dioxide molecule polarizes the carbon atom making it more susceptible to nucleophilic attack Nucleophilic Attack The Zn bound OH - attacks the carbon of CO 2 to form HCO 3 -
Carbonate ion is formed. Carbonate is removed from complex. See figure to see where it goes HCO 3 - Dissociation A water molecule replaces the HCO 3 - product. This happens simultaneously as the bicarbonate ion dissociates. The water is complexed in the Zn Threonine environment. The proton is therefore very acidic and is transferred to a water molecule bound to His 64
H + Dissociation/Shuttle The proton product dissociates from the water / Zn complex and is transferred in three steps along a "wire" of H-bonded waters to His 64. These are Proton transfer down His 64 chain
His 64 Rotation/Flip The protonated His 64 side chain rotates from the "in" postion to the "out" position where it is exposed to bulk solvent on the enzyme exterior. H+ dissociation. This the rate limiting step in the reaction is H + dissociation.
His rotates back in. Enzyme is ready to repeat sequence. Reaction mechanism and notes from: http://info.bio.cmu.edu/courses/03231/protstruc/bctmech.htm#bctmech. This site is highly recommended as it contains an animation of the reaction sequence and an interactive applet that permits detailed investigation of the enzyme. Figure 8a. The Histidine Swing http://www.rcsb.org/pdb/molecules/pdb49_3.html
Figure 9. Use of carbonic anhydrase inhibitors Categories Altitude sickness, acute, prophylactic and therapeutic agent Acetazolamide Anticonvulsant Acetazolamide Antiglaucoma agent, systemic Acetazolamide Dichlorphenamide Methazolamide Antiparalytic, familial periodic paralysis Acetazolamide Antiurolithic, cystine calculi Acetazolamide Antiurolithic, uric acid calculi Acetazolamide Diuretic, urinary alkalinizing Acetazolamide http://www.swedish.org/15672.cfm