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Important terms of enzymology Course 2 / 210 Vladimíra Kvasnicová Biochemical reactions are catalyzed by enzymes: Enzymes are commonly called according to the: a) type of the chemical reaction b) type of the substrate The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006) ENZYMES Laboratory order form from http://spch.cz/kliniky/kbi/laboratorni_prirucka/zadanka_biochemie.pdf (December 2006) „ Cardiac enzymes“ The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2 abbreviations of enzymes • used in a medicine • e.g. LD, ALT, ALP old trivial names • no relationship to the catalyzed reaction • suffixed by -in (pepsin, trypsin) • used for long time known enzymes Enzyme nomenclature EC nomenclature http://www.chem.qmul.ac.uk/iubmb/enzyme/ * each enzyme is classified by EC number (Enzyme Commission of IUBMB) – 6 classes: • • • • • • EC 1.x.x.x EC 2.x.x.x EC 3.x.x.x EC 4.x.x.x EC 5.x.x.x EC 6.x.x.x oxidoreductases transferases hydrolases lyases isomerases ligases (synthetases) → classification by a reaction catalyzed by the enzyme example: IUBMB Enzyme Nomenclature EC 2.7.1.1 Accepted name: hexokinase Reaction: ATP + D-hexose = ADP + D-hexose 6-phosphate Other name(s): hexokinase type IV glucokinase; hexokinase D; hexokinase type IV; hexokinase (phosphorylating); ATP-dependent hexokinase; glucose ATP phosphotransferase Systematic name: ATP:D-hexose 6-phosphotransferase Comments: D-Glucose, D-mannose, D-fructose, sorbitol and Dglucosamine can act as acceptors; ITP and dATP can act as donors. The liver isoenzyme has sometimes been called glucokinase. The reference is found at http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/1/2.html systematic names • are made according to special rules, they specify a reaction catalyzed by the enzyme example: ATP : D-glucose phosphotransferase (EC 2.7.1.2) → transfers (2) phosphate (7) to an alcohol group (1) ATP + D-Glc → ADP + D-Glc-6-phosphate (Glc-6-P) EC 2.7.1.2. = glucokinase = accepted name oxidation / reduction (ions, organic compounds) enzymes: oxidoreductases (EC 1.x.x.x) distinguish: dissociation / pH / acidity / H+ / H30+ dehydrogenation / H / H2 • „redox equivalents“ FADH2 NADH+H+ NADPH+H+ FAD (oxidized) FADH2 (reduced) http://biochem.siuc.edu/web_lessons/bmb_vit.htm http://dolly.biochem.arizona.edu/Bioc462b_Honors_Spring_2009/kyang/whatisnad2.html common names (= accepted names) * • simple, commonly used in practice • very important! 1. oxidoreductases: Aox + Bred → Ared + Box * dehydrogenase (H- or H) * reductase * oxidase * peroxidase (various peroxides) * oxygenase (O2) * hydroxylase (= monoxygenase; -OH) * desaturase (-CH2CH2- → -CH=CH-) enzymes transferring groups = transferases (EC 2.x.x.x) → transfer of –NH2, phosphate, acyl, C1-fragments,... distinguish: acyl of an acid anion of an acid 2. transferases: A-x + B → A + B-x * grouptransferase (e.g. aminotransferase) * kinase (= phosphotransferase) * phosphorylase * transketolase * transaldolase enzymes catalyzing hydrolysis = • reactions: 3. hydrolases: hydrolases (EC 3.x.x.x) condensation / hydrolysis A-B + H2O → A-H + B-OH „substrate“-ase * peptidase, glycosidase, lipase, nuclease * esterase (R1-CO-O-R2) (phosphate-O-R) → Pi !!! * phosphatase * phosphodiesterase (R1-O-phosphate-O-R2) enzymes catalyzing introduction or releasing of a small compound = lyases (EC 4.x.x.x) - addition or elimination of water = hydration / dehydration - removing of CO2 = decarboxylation (not carboxylation!) 4. lyases: A-x ↔ B + x * decarboxylase (→ CO2) * dehydratase (→ H2O) * hydratase (-CH=CH- + H2O → -CH(OH)CH2-) * (synthase) enzymes catalyzing isomerization = isomerases (EC 5.x.x.x) isomers = chemicals having the same molecular formula but differing in their structures e.g. C6H12O6 5. isomerases: glucose / fructose A → iso-A * epimerase (monosacharide → its epimer) * mutase (rearangement of a phosphate group) enzymes catalyzing synthetic reaction which needs energy from an energy rich compound = 6. ligases: ligases (EC 6.x.x.x) A + B + ATP → A-B + ADP + Pi * carboxylase * (synthetase) examples: • pyruvate carboxylase • glutamine synthetase = glutamate-ammonia ligase Add class to which each enzyme belongs AST ALT GMT ALP ACP AMS LPS CK CHE LD aspartate aminotransferase alanine aminotransferase gamma-glutamyl transpeptidase alkaline phosphatase acid phosphatase α-amylase lipase creatine kinase cholinesterase lactate dehydrogenase Cofactors of oxidoreductases: NAD+ nicotinamide adenine dinucleotide NADP+ nicotinamide aden. dinucl. phosphate (precursor: niacin = nicotinic acid) HFAD FMN flavin adenine dinucleotide flavin mononucleotide 2H (precurzor: riboflavin = vitamin B2) heme Fe3+ + e- ↔ Fe2+ ⇒ e- transferases: ATP adenosine triphosphate / phosphate GTP guanosine triphosphate / phosphate TDP thiamine diphosphate / C-fragment (prekurzor: thiamine = vitamin B1) PALP pyridoxal phosphate / -NH2 (prekurzor: pyridoxine = vitamin B6) THF tetrahydrofolate / C1-fragment (prekurzor: folic acid) CoA coenzyme A (HS-Co-A) / acyl PAPS phosphoadenosine phosphosulfate / sulfate 3´-phosphoadenosine-5´-phosphosulfate (PAPS) transfers sulfate group to a substrate (sulfatation) http://web.indstate.edu/thcme/mwking/amino-acid-metabolism.html (Jan 2007) Coenzyme A = CoA-SH http://lxyang.myweb.uga.edu/bcmb8010/pic/NAD+.gif and http://oregonstate.edu/instruct/bb450/stryer/ch14/Slide26.jpg (Jan 2008) Derivates of tetrahydrofolate http://www.dentistry.leeds.ac.uk/biochem/postgrad/thftypes.gif (Jan 2008) lyases: PALP ligases: ATP biotin pyridoxal phosphate (decarboxylases) adenosine triphosphate → acyl-CoA-synthetases → aminoacyl-tRNA-synthetases = vitamin H (carboxylases) Enzymes • • • • • • lower an energy of activation (EA) reduce the time to reach the reaction equilibrium are not consumed or changed by the reaction help the reaction proceed under a body´s T, p and pH are specific can be regulated • don´t change the ∆G of the reaction • don´t change the equilibrium position of the reaction self study The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2 Each enzyme has temperature optimum pH optimum affinity to its substrate The figure is found at: http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt113/05jpeg/05_jpeg_HTML/index.htm (December 2006) Some enzymes are produced as precursors (= PROENZYMES or ZYMOGENS) The figure is found at: http://wine1.sb.fsu.edu/bch4053/Lecture26/zymogen.jpg (December 2006) or must be activated to be active (e.g. by phosphorylation): The figure is found at: http://fig.cox.miami.edu/~cmallery/150/memb/c11x11enzyme-cascade.jpg (December 2006) Isoenzymes (isozymes) are enzymes which catalyze the same reaction but differ in their primary structure and phyzico chemical properties Isoenzymes are • produced by different genes (= true isozymes) • or produced by different posttranslational modification (= isoforms) • found in different compartments of a cell • found in different tissues of an organism • can be oligomers of various subunits (monomers) example: 5 isozymes (various monomer ratio) The figure is found at: http://wine1.sb.fsu.edu/bch4053/Lecture26/isozymes.jpg (December 2006) separate enzymes of a mtb pathway multienzyme complexes This is Figure 17.6 from Garrett, R.H.; Grisham, C.M. Biochemistry; Saunders: Orlando,1995; page 553, found at http://www.uwsp.edu/chemistry/tzamis/enzyme_complex.html (December 2006) example: 2-oxoacid dehydrogenase multienzyme complex The figure is found at: http://faculty.uca.edu/~johnc/pdhrxns.gif (December 2006) Allosteric enzyme: a) monomeric, b) oligomeric The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2 Allosteric enzyme in T and R conformations: modulators shift the equilibrium inhibitors have a greater affinity for T-state activators and substrates have a greater affinity for R-state The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2 Determination of enzyme activity for diagnostic purposes most often blood is investigated (serum, plasma) → evaluation of presence and seriousness of a tissue damage units: µkat/L (= catalytical concentration of enzyme) kat = katal 1 katal = 1 mole of a substrate transformed per 1 sec 1 µkat = 10-6 kat Enzymes found in plasma: a) plasma-specific enzymes (e.g. clotting factors) b) secretory enzymes (e.g. digestive enzymes) c) cellular enzymes Important knowledge: 1) intracellular localization of enzymes 2) organ and tissue distribution of enzymes 3) sources of enzymes found in plasma 4) way of enzyme elimination from blood Enzyme kinetics • activity, units 1 katal = 1 mole of a substrate transformed per 1 sec 1 IU = 1 µmole of a substrate transformed per 1 minute 1 katal 1 katal = 1 mole = 106 µmole = 60 x 106 µmole = 6 x 107 IU / / / 1 sec 1 sec 1 min (= 60 sec) The activity is related to a constant concentration of an enzyme: [E] = constant The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006) ! REMEMBER ! The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/gk3x15.gif (December 2006) Michaelis-Menten kinetics • the curve can be described by the equation: The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006) Km describes an affinity of the enzyme to its substrate ! indirect proportionality! The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/gk3x15.gif (December 2006) linearization of the curve: y = kx + q The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006) The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/gk3x15.gif (December 2006) Inhibition of enzymes The figure is found at: http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt113/05jpeg/05_jpeg_HTML/index.htm (December 2006) 1) Competitive inhibition • inhibitor resembles substrate • it is bound to an active site but not converted by the enzyme • increases Km (↓afinity of enzyme to its S) • if concentration of a substrate is increased the inhibition is decreased • the inhibition is reversible The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006) 2) Noncompetitive inhibition • inhibitor binds at a site other than the substrate-binding site • inhibition is not reversed by increasing concentration of substrate (no Km change) • Vmax is decreased (it is related to decreasing of active enzyme concentration) • reversible only if the inhibitor is not bound by covalent bond The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006) Summary of the inhibition The figure is found at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html (December 2006) Some enzymes can be inhibited also by an excess of their substrate The figure is found at: http://www-biol.paisley.ac.uk/Kinetics/chapter_3/chapter3_6_1.html (December 2006) Inhibition by drugs and poisons a) reversible b) irreversible → inhibitor is bound covalently into the active site of enzyme Inhibition as a regulation of metabolic pathways: inhibition by products or intermediates: a) feedback regulation b) cross-regulation c) feedforword regulation inhibition by d) reversible covalent modification (e.g. phosphorylation / dephosphorylation) Reversible covalent modification: A) • phosphorylation by a protein kinase • dephosphorylation by a protein phosphatase B) • phosphorylated enzyme is either active or inactive (different enzymes are influenced differently) The figure is found at: http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt113/05jpeg/05_jpeg_HTML/index.htm (December 2006) Inhibition of enzymes used in the regulation is either • competitive (Km is increased above substrate concentration within a cell) or • allosteric (by conformational changes affecting the catalytic site) Allosteric regulation • activator is „a positive modulator“ • inhibitor is „a negative modulator“ ! the curve of allosteric enzymes is sigmoidal not hyperbolic ! The figure is found at: http://www-biol.paisley.ac.uk/Kinetics/Chapter_5/chapter5_2_2.html (December 2006) SUMMARY Regulation of enzyme activity • • • • • • availability of a substrate and its concentration induction of synthesis of a regulatory enzyme activation of enzyme precursors covalent modification of enzymes competitive inhibition allosteric regulation