‹#› 1 Enzymes II Ó Department of Biochemistry (J.D.) 2013 Mechanism of enzyme reaction, metalloenzymes, kinetics, activity, enzymes in medicine ‹#› 2 Active site of enzyme •small part of molecule, deep cleft or pocket on the surface •three-dimensional environment shields the substrate from water •in oligomeric enzymes - interface between subunits •made by side chains of AA which are distant in primary structure •the site is really active - protein flexibility facilitates the conformation changes fitting to substrate •substrate is bound relatively weakly ‹#› 3 Binding substrate to enzyme •binding a substrate induces the conformational changes in enzyme molecule (induced fit model) •an enzyme-substrate (ES) complex is formed ‹#› 4 A deep, relatively hydrophobic pocket At the bottom of the deep pocket there is an acidic residue (Asp) Two residues of valine close off the mouth of the small hydrophobic pocket Example: The active sites of three proteinases Certain AA residues determine the substrate specificity of these enzymes. The peptide bonds preferably hydrolyzed: by chymotrypsin - near residues with large, hydrophobic, uncharged side chains (Phe, Trp), by trypsin – near residues with long, positively charged side chains (Arg, Lys), by elastase - near residues with small hydrophobic side chains (Gly, Ala). ‹#› 5 Catalytic mechanism depends on the number of substrates •Monosubstrate reactions • S + E D { ES.......EP } D P + E • •Bisubstrate reactions (more common) • SA + SB D { ESAB.......EPAB } D PA + PB + E • • Sequential reaction: both substrates bind to enzyme, chemical conversion proceeds, and both products are released • Ping-pong reaction: typical for aminotransferases, one substrate is attached, converted, and released, • then the same events occur with the second substrate ‹#› 6 Sequential reaction: - type ordered - the substrates bind the enzyme in a defined sequence - type random – the order of addition of substrates and release of products is random: Ping-pong reaction: + + + ‹#› 7 Catalytic groups •located in active site •involved the chemical conversion of substrate •nucleophilic (cysteine -SH, serine -OH) •acidic (Asp, Glu), basic (His, Arg, Lys) Examples of different types of catalytic mechanisms: • Catalysis through proximity and orientation effects (strained reactants) • Covalent catalysis – formation of transitory covalent bonds between enzyme and substrate • Acid-base catalysis – protonization of substrates or catalytic groups of enzyme • Metal ion catalysis mediating redox reactions or shielding negative el. charges • Electrostatic catalysis (after excluding water from the active site by binding a substrate) ‹#› 8 SERINE PROTEASES Serine 195 Chymotrypsin, trypsin CYSTEINE PROTEINASES Carboxypeptidase A, thermolysin (bacterial) METALLOPROTEINASES Cathepsins, papain ASPARTATE PROTEINASES Pepsin, renin Examples of proteinases Due to the different arrangement of active sites, they exhibit • different substrate specificities • different catalytic mechanisms ‹#› 9 Example: active site of chymotrypsin Nucleophilic attack of serine -OH to carbonyl carbon of peptide bond Þ serine protease 195 57 simplified ‹#› 10 Active site of chymotrypsin: the cleavage of peptide bond ‹#› 11 Metalloenzymes •contain functioning metal cations (prosthetic groups) directly involved in catalyzed reaction, metal ions attached tightly (Enz-M) •some enzymes need metal ions just for activation, they are associated relatively weekly (Enz …M), e.g. Ca2+ (coagulation factors), Mg2+ (kinases) • • ‹#› 12 Metal ion is a part of ternary complex •three components make a ternary complex: enzyme (Enz), substrate (S), and metal ion (M) •different types of complexes: Enz-S-M, Enz-M-S, or cyclic complexes •vacant metal orbitals accept electron pair of nucleophil to make s-bond •metal ions make chelates with some groups of enzyme/substrate Þ deformation of structure Þ strain Þ facilitates chemical conversion •coordination sphere of metal creates a three-dimensional template Þ stereospecific control of reaction possible mechanism ‹#› 13 Molybdenum (Mo) •Some oxidoreductases •cofactor – molybdopterin •Xanthine oxidase (xanthine ® uric acid) •Sulfite oxidase (sulfite HSO3- ® sulfate SO42-) • Mo in food: legumes, wholemeal cereals ‹#› 14 Zinc (Zn) •Many enzymes •Alcohol dehydrogenase (ethanol ® acetaldehyde) •Carbonic anhydrase (H2O + CO2 D H2CO3) •Carboxypeptidases (cleavage of polypeptides from C-terminal) •Cu, Zn-superoxide dismutase (cytosolic isoform) (2 •O2- + 2 H+ ® O2 + H2O2) • Zn in food: red meat, lobsters, legumes, seeds, wholemeal cereals ‹#› 15 Copper (Cu) •many oxidoreductases •Ceruloplasmin (ferroxidase) (Fe2+ ® Fe3+) •Cytochrome-c-oxidase (R.Ch., electron transfer to O2) •Monoamine oxidases (MAO, inactivation of biogenic amines, side product H2O2, see Med. Chem. II, p. 60) •Dopamine hydroxylase (dopamine ® noradrenaline) •Lysyl oxidase (collagen maturation, Lys ® alLys) Cu in food: liver, meat, cocoa, legumes, nuts ‹#› 16 Manganese (Mn) •some hydrolases, decarboxylases, transferases •Mn-superoxide dismutase (mitochondrial isoform) •Arginase (arginine ® urea + ornithine) •Synthesis of proteoglycans + glycoproteins • Mn in food: legumes, nuts, wholemeal cereals ‹#› 17 Iron (Fe) •heme and non-heme iron enzymes •Catalase (heme, H2O2 ® H2O + ½O2) •Myeloperoxidase (heme, neutrophils) H2O2 + Cl- + H+ ® HClO + H2O •Cytochromes (heme, transfer of electrons in R.Ch.) •Fe-S proteins (non-heme, transfer of electrons in R.Ch.) Fe in food: animal blood, red meat, liver, egg yolk, nuts, broccoli ‹#› 18 Selenium (Se) •few enzymes (redox reactions), Se in selenocysteine •Glutathione peroxidase (2 GSH + H2O2 ® 2 H2O + G-S-S-G) •Deiodases of thyronines (thyroxine T4 ® triiodothyronine T3) •Thioredoxin reductase (ribose ® deoxyribose) Se in food: sea products, legumes ‹#› 19 Factors determining the rate of enzymatic reaction Temperature Reaction rate increases with temperature, optimal around 40 °C, at higher temperatures the rate decreases due to enzyme denaturation pH pH value determines ionization of groups in active site and its surroundings, Constant pH of body fluids is maintained by buffer systems Each enzyme has its pH optimum, intracellular enzymes around pH 7 Digestion enzymes differ: pepsin pH 2 Activators They accelerate enzyme reaction Often divalent metal cations: Ca2+, Mg2+, Zn2+, Mn2+ Inhibitors Slow down or stop enzyme reaction competitive: similar to substrate, they compete for active site Other, e.g. heavy metal ions, strongly bind to important enzyme groups Substrate concentration At high substrate concentration, enzyme is saturated, so the reactions proceeds with maximal velocity, graphically expressed as saturation curve ‹#› 20 Basic kinetic terms •reaction: S ® P (S = substrate, P = product) •definition of reaction velocity (rate): • this definiton is for average rate, instantaneous rate: d[S]/dt ‹#› 21 Kinetic equation for reaction S ® P v = k [S] = k [S]1 Þ reaction of 1. order k = rate constant ‹#› 22 Kinetic (progress) curves for substrate and product [P] t [S] t (equilibrium) during reaction: concentration of substrate decreases concentration of product increases reaction rate is determined from kinetic curves The instantaneous velocity vx at any particular time tx is given by the slope of the tangent to the curve at that time. ‹#› 23 Reaction of 0. order •Reaction rate does not depend on the substrate concentration •v = k [S]0 = k × 1 = k = constant •At great excess of substrate S in enzyme rections only in laboratory conditions ‹#› 24 Initial velocity vo •The highest value of velocity •It is not influenced by the decrease of substrate nor the reverse change of product •Determined from kinetic curves at the time t = 0 ‹#› 25 Initial velocity vo depends on substrate concentration •Michaelis-Menten equation •for one-substrate reactions • Vmax = maximal velocity (for the given concentration of enzyme) Km = Michaelis constant ‹#› 26 The graph of previous equation is saturation curve enzyme saturated by substrate ‹#› 27 If [S] << Km at low substrate concentration, the reaction proceeds by the 1st order kinetics × ‹#› 28 If [S] >> Km at high substrate concentration, the reaction proceeds by the 0. order kinetics × ‹#› 29 Two parts of saturation curve compare with pages 21 and 23 [S] V0 Vmax Km The zero-order kinetics The 1st order kinetics ‹#› 30 If [S] = Km ‹#› 31 Significance of Km and Vmax • the Michaelis constant Km is the concentration of substrate [S] which gives half the maximal velocity Vmax (50 % saturation of enzyme) • the Km has the dimension of concentration (mol/l) • Km is inversely related to the affinity of enzyme for its substrate. If more substrates with similar structure exist, then the best natural substrate is one with the least value of Km • if there is a need to measure the activity of enzyme, the substrate concentration has to be at least several times higher than the Km value. ‹#› 32 [P] t [S1] [S2] [S3] [S4] v0 1 v0 2 v0 3 v0 4 From the obtained progress curves, the values vo are estimated and plotted against the corresponding [S]. The velocity vo rises linearly as substrate concentration increases, and then begins to level till it reaches a limit value at high substrate concentrations. How to get a saturation curve? [E] = const. A series of measurements of initial velocity is arranged at a constant enzyme concentration [E] and different substrate concentrations [S] (in the range of 2 - 3 orders). ‹#› 33 Distinguish •Kinetic curve •time record of one reaction •[S] = f (t) or [P] = f (t) •Saturation curve •dependence obtained from many reactions (see previous page) •vo = f ([S]) [S] …….. substrate concentration [P] .......... product concentration f ………… function t ………… time vo ……….. initial velocity ‹#› 34 Vmax and Km describe the kinetic properties of enzyme •are hardly obtained from saturation curve •easily obtained from linear double reciprocal plot •Lineweaver-Burk: 1/vo is plotted against 1/[S] ‹#› 35 Reciprocal equation ‹#› 36 Reciprocal form is the equation of a line (y = a x + b) 1/vo ................ dependent variable (y) 1/[S] ............... independent variable (x) 1/Km ............... 1/Vmax ............. easily determined from the graph ‹#› 37 Linear reciprocal plot: 1/vo is the function of 1/[S] ‹#› 38 vo [S] [E1] [E2] > [E1] [E3] > [E2] KM does not change, Vmax increases with increased [E] Initial velocity depends on enzyme concentration [E] saturated enzyme: vo = k [E]t [E]t is total concentration of enzyme • ‹#› 39 How to quantify enzymes in biological material? • very low concentrations of enzymes • in very complex mixtures • in the presence of many other proteins • simple chemical reactions cannot be used, they are not specific enough to distinguish individual enzymes ‹#› 40 Enzyme quantity in a biological sample can be determined by two ways • •catalytic concentration •μkat/l •the product of enzyme reaction is determined • • •mass concentration •μg/l •enzyme itself is determined as antigen (immunochemical assay) ‹#› 41 Catalytic activity of enzyme •unit katal, 1 kat = mol/s •such amount of enzyme that catalyzes the conversion of one mole of substrate per one second IU (international unit), 1 IU = μmol/min 1 μkat = 60 IU, 1 IU = 16.6 nkat Catalytic concentration of enzyme •activity related to the volume of body fluid (blood serum) •typical units: mkat/l, mkat/l • ‹#› 42 Distinguish: unit and its dimension Quantity Unit Dimension Catalytic activity kat mol/s Catalytic concentration kat/l mol/l.s ‹#› 43 Determination of catalytic activity of ALT: two coupled enzyme reactions alanine + 2-oxoglutarate ® pyruvate + glutamate blood serum sample (ALT) pyridoxal-P pyruvate + NADH+H+ ® lactate + NAD+ ΔA/Δt optical (UV) test Seminars, p.18 the decrease of absorbance at 340 nm is followed in time LD ‹#› 44 Determination of catalytic activity in vitro •optimal conditions: temperature, pH the presence of all necessary cofactors the absence of all (known) inhibitors •excess of substrate Þ 0. order kinetics: [S] >> Km Þ saturated enzyme, reaction rate is constant and close to Vmax •D[S] or D[P] is followed during time ‹#› 45 Two methods for catalytic concentration Feature Kinetic method Constant-time method What is measured [S] or [P] [P] How continually (e.g. in 10 seconds) after some time (e.g. 10 min) the reaction is stopped by enzyme inactivation Kinetic curve needed yes no What is determined initial velocity vo average velocity Evaluation of method exact less exact ‹#› 46 Problem 1 Enzyme sample (0.1 ml) was added to substrate solution. After 5 min, 0.2 mmol of product was determined. What is catalytic concentration of enzyme? ‹#› 47 Problem 1 - Solution = 6,7 mkat/l ‹#› 48 Problem 2 Reaction mixture contains: 2.5 ml buffer 0.2 ml solution of NADH (optical UV test) 0.1 ml blood serum 0.2 ml substrate solution After 60 s, the decrease of NADH absorbance is DA = 0.03 eNADH = 6220 l/mol.cm, cuvette width l = 1 cm. What is catalytic concentration of enzyme? ‹#› 49 Problem 2 - Solution Serum sample was diluted: Vfinal/Vinitial = 3,0 / 0,1 = 30 Lambert-Beer law: DA = e Dc l changes of absorbance and concentration expressed per time Dt Þ DA/Dt = e Dc l/Dt Dt = 60 s Multiplied by dilution: 30 × 8 × 10-8 = 2,4 × 10-6 mol/l.s = 2,4 × 10-6 kat/l = 2,4 mkat/l ‹#› 50 Consider that catalytic concentration = rate of chemical reaction kat/l = mol/l.s ‹#› 51 •Irreversible inhibitors •Irreversible inhibitors are usually compounds not of biological origin, •bind onto enzyme mostly covalently and make substrate binding impossible. •Heavy metal ions, organophosphates, cyanides •bind and inhibit irreversibly enzymes during isolation. Inhibitors reduce enzyme activity Reversible inhibitors In contrast with irreversible inhibitors, reversible inhibitors bind to the enzyme loosely and can rapidly dissociate from the enzyme-inhibitor complex. These inhibitors are classified as competitive, non-competitive. ‹#› 52 Example of irreversible inhibition Diisopropyl fluorophosphate (and similar pesticides and nerve gases) inhibits acetylcholine esterase by phosphorylation of a crucial serine residue. ‹#› 53 • resemble the substrates (similar shape of molecule) • bind to the active sites, but the complex is non-reactive • they compete with normal substrates for the active sites Competitive inhibitors ‹#› 54 Malonate competitively inhibit succinate dehydrogenase Examples Methotrexate Tetrahydrofolate Methotrexate competitively inhibits active sites for tetrahydrofolate of the dihydrofolate reductase in the synthesis of purine and pyrimidine bases of nucleic acids. It is used to treat cancer. Malonate Succinate COO– CH2 CH2 COO– COO– CH2 COO– ‹#› 55 Methanol poisoning is treated by ethanol ethanol and methanol are similar molecules they compete for active site in enzyme alcohol dehydrogenase ‹#› 56 Km [S] v0 Vmax Km 1 / v0 1 / [S] 1 / Vmax - 1 / Km No inhibitor Competitive inhibitor Competitive inhibitors increase Km without any change in Vmax The Vmax can be reached even in the presence of inhibitor, but at much higher concentrations of [S] that have to overcome the competing inhibitor concentration. ‹#› 57 Non-competitive inhibition Non-competitive inhibitors bind to both free enzyme and enzyme-substrate complex, but in contrast to competitive inhibitors, not in the active site (the structure of inhibitor is distinct from that of substrate). Non-competitive inhibition cannot be overcome by increasing the substrate concentration. The non-inhibited remaining molecules of the enzyme behave like a more diluted solution of the enzyme. ‹#› 58 1/v o 1 / [S] 1 / Vmax - 1 / Km Non-competitive inhibitor No inhibitor Km [S] v0 Vmax Vmax Non-competitive inhibitor Non-competitive inhibitors decrease Vmax without any change in Km ‹#› 59 Many drugs are inhibitors of enzymes •Acetylsalicylic acid, ibuprofen (cyclooxygenase) - see Seminars, p. 67 •Statins (HMG-CoA reductase) inhibit cholesterol synthesis (e.g. lovastatin) •ACE Inhibitors (angiotensin-converting enzyme) – treatment of hypertension •Reversible acetylcholinesterase inhibitors (e.g. neostigmine) – myastenia gravis, post-surgical atonia, •Brain acetylcholinesterase inhibitors (rivastigmine, galantamin) – Alzheimer d. •Many antibiotics inhibit bacterial enzymes: • Penicillins – inhibit bacterial transpeptidases (formation of bacterial cell-wall) • Tetracyclins, macrolides, chloramphenicol – inhibit bacterial proteosynthesis • Fluoroquinolones (ciprofloxacin) – inhibit bacterial topoisomerase ‹#› 60 1.Regulation of enzyme quantity 2.Regulation of enzyme activity 3.Availability and concentration of substrate and/or cofactor (in vivo less important, in vitro critical condition) 4. Regulation principles in enzyme reactions (three general aspects) ‹#› 61 Regulation of enzyme quantity •Controlled enzyme proteosynthesis constitutive and induced gene expresion, regulation of transcription rate, posttranscriptional RNA processing, regulation of translation rate and posttranslational modifications • •Controlled enzyme degradation specific intracellular proteinases determine biological half-life of enzymes ‹#› 62 Regulation of enzyme activity •Activation by partial and irreversible proteolysis •Reversible covalent modification •Allosteric regulation • • ‹#› 63 Enzyme activation by partial proteolysis •Active enzyme is formed by irreversible cleavage of certain sequence from proenzyme (zymogen) molecule •Proteinases in digestion (pepsinogen ® pepsin) •Factors of blood coagulation •Proteinases caspases in apoptosis ‹#› 64 Reversible covalent enzyme modification •phosphorylation, catalyzed by kinases •transfer of phosphoryl -PO32- from ATP to -OH group of enzyme (Ser, Thr, Tyr) •reversible process, dephosphorylation catalyzed by phosphatase, hydrolysis of phosphoester •Other modifications: carboxylation, acetylation, prenylation ... ‹#› 65 Phosphorylation and dephosphorylation of enzyme alters its activity protein kinase phosphoprotein phosphatase protein-serine-OH protein-serine protein kinases are activated by second messenger (cAMP, calcium calmodulin complex) ‹#› 66 The consequences of phosphorylation/dephosphorylation in two antagonistic enzymes Feature Glycogen phosphorylase Glycogen synthase Enzyme catalyzes glycogen degradation by energy-poor phosphate (Pi) glycogen synthesis from energy-rich UDP-glucose By phosphorylation, enzyme is activated inhibited By dephosphorylation, enzyme is inhibited activated ‹#› 67 Allosteric enzymes are oligomeric •more subunits, often regulatory and catalytic •effector, structurally different from substrate - often product, binds to enzyme to allosteric site (other than active site) •binding effector triggers the changes in conformation and activity ® allosteric activation / inhibition ‹#› 68 Allosteric enzymes are oligomeric allosteric enzyme ‹#› 69 Saturation curve of allosteric enzymes is sigmoidal bez efektoru activation without effector inhibition ‹#› 70 Cooperative effect •in oligomeric enzymes and non-catalyzing proteins (e.g. Hb) •more subunits = more active sites •binding substrate (or O2 to Hb) to one subunit/active site induces conformation changes in other subunits/active sites so that other substrate (or O2) molecules bind more easily (or more hardly) •example: hemoglobin (tetramer) × myoglobin (monomer) • ‹#› 71 Compare Saturation of enzyme by substrate Saturation of hemoglobin by oxygen vo [S] pO2 Saturation (%) myoglobin (monomer) hemoglobin (tetramer) allosteric enzyme (oligomeric) simple enzyme (complies with M.-M. kinetics) ‹#› 72 Three utilizations of enzymes in medicine 1.enzymes as indicators of pathological condition 2.enzymes as analytic reagents in clinical chemistry 3.enzymes as drugs ‹#› 73 Examples of enzymes in clinical diagnostics Enzyme Reference values Elevation in serum indicates ALT CK PSA up to 0,9 mkat/l up to 4 mkat/l up to 4 μg/l hepatopaties myopaties, myocardial infarction prostate cancer ALT alanine aminotransferase, CK creatine kinase, PSA prostate specific antigen In cell damages, activity of intracellular enzymes in extracellular fluid (blood serum) is elevated ‹#› 74 Creatine kinase (CK) is a dimer and makes three isoenzymes Isoenzyme Organ % of total activity in blood plasma Elevated value CK-MM CK-MB CK-BB muscles heart brain 94-96 % up to 6 % traces muscle trauma infarction brain damage •catalyze the same reaction •differ in primary structure, physical, and kinetic properties •often have different tissue distribution •determined mostly by electrophoresis and/or by immunochemical assays • Isoenzymes/Isoforms ‹#› 75 Enzymes as analytic reagents Enzyme Enzyme origin Assay for Glucose oxidase Peroxidase Lipase Cholesterol oxidase Uricase Bilirubin oxidase Urease Lactate dehydrogenase Taq polymerase Aspergillus niger horse radish (Armoracia sp.) Candida sp. Pseudomonas sp. Candida sp. Myrothecium sp. Canavalia sp. Pediocus sp. Thermus aquaticus glucose glucose triacylglycerols cholesterol uric acid bilirubin urea ALT, AST PCR method ‹#› 76 Enzymatic determination of glucose glucose + O2 ¾¾® gluconolactone + H2O2 H2O2 + H2A ¾¾® 2 H2O + A glucose oxidase peroxidase colourless chromogen coloured product (absorbance measured) The principle of glucose determination in biochemical analyzers and in personal glucometers ‹#› 77 Glucometer Personal glucometer (Practice in 4. semester) ‹#› 78 Pancreatic enzymes in therapy •enzyme mixtures (lipase, amylase, proteinases) of animal origin •indication: insufficient secretion of pancreas, cystic fibrosis •3 × times daily a capsule during meal • gastro-resistant capsules, they survive the passage through stomach, soluble and active in duodenum ‹#› 79 Asparaginase in leukemia treatment •Catalyzes the hydrolysis of asparagine amide group (deamidation) •Asn + H2O ® Asp + NH3 •L-asparagine is necessary for the proteosynthesis of some cancer cells •Hydrolysis of Asp reduces the cell proliferation (see also Seminars, p. 19) • Enzyme fibrinolytics •thrombolytic drugs, dissolve blood clots in veins •urokinase (human enzyme, serine protease) •converts plasminogen to plasmin ® which degrades fibrin ® thrombolysis •venous thrombosis, pulmonary embolism, acute myocardial infarction ‹#› 80 Proteases in enzyme therapy •Local treatment •fibrinolysin, chymotrypsin, collagenase and other •degrade necrotic tissue, clean wounds, decubital ulcers etc. •Systemic treatment •trypsin, chymotrypsin, papain (papaya), bromelain (pineapple) •anti-inflammatory agents •sports injury, trauma, arthritis, other kinds of swelling, arthritis etc. •Wobenzym, Phlogenzym and other ananas papaja