CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 3ENZYME: Lecture Handout I.General Description of Enzymes A.Chemical composition 1.Enzymes are proteins, compounds of high molecular weight, containingCarbon (C), hydrogen (H), oxygen (O), sulfur (S), nitrogen (N) 2.Possess primary, secondary, tertiary (sometimes quaternary) structure 3.Same properties as other proteins a.Charge (isoelectric point, electrophoresis) b.Solubility c.Denaturation (extremes in heat, pH, chemical) d.Non-specific or specific (antigenic) binding 4.Some enzymes have iosenzyme forms: a.Different form of an enzyme due to differing arrangements of thepolypeptide units in the quaternary structure b.Maintains specificity for the same chemical reaction c.Possess unique diagnostic value: show specificity to certain organsystems 1)Isoenzymes of creatine kinase (CK) CK-MM to skeletal muscle CK-MB to cardiac tissue 2)Isoenzymes of lactate dehydrogenase (LD) LD1 and LD2 to cardiac tissue LD4 and LD5 to liver and skeletal muscle B.Biochemical Function 1.Catalyze chemical reactions: accelerates specific chemical reactionstoward equilibrium S P Substrate (S) + Enzyme (E) ESProduct (P) + Enzyme 2.Lowers the energy of activationrequired for the reaction to proceed CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 43.Properties of enzymes a.Effective in small concentrations b.Remain unchanged in the reaction, not consumed in the reaction c.Effect speed of reaction (not final concentration of substrate orproducts) d.Show greater degree of specificity than chemical catalysts e.Unstable and easily destroyed 4.Cofactors: substances associated with some enzymes that are needed formaximal enzyme activity a.Coenzyme: organic cofactors (NAD, NADH); serve as secondsubstrates for enzyme reactions b.Activators: inorganic cofactors (Ca++, Fe++, Zn++, K+, Mg++) C.Importance of Enzymes in Health and Disease 1.Enzymes are intracellular 2.Enzyme concentration differs dependent upon type of tissue a.AST: high concentration in liver b.Amylase: high concentration in pancreas and salivary glands 3.Normal enzyme levels (measured in plasma) reflect normal cell turnover 4.Increased levels of enzymes in plasma a result of cellular/tissue/organdamage (necrosis)orincreased synthesis a.AST: hepatitis, liver disease b.Amylase: pancreatitis or mumps 5.Decreased levels of enzymes usually inherited deficiency: inborn error ofmetabolism a.PKU disease b.Galactosemia c.Lipid storage disorders 6.Isoenzyme forms of an enzyme often show organ specificity a.CK-MB increased in acute myocardial infarction (AMI) b.LD1 and LD2 increased in hemolytic anemia, AMI D.Specificity of the enzyme to substrate 1.Substrate molecule attaches to a specialized region on the enzyme a.Unique sequence/orientation of amino acids forming a groove orpocket on its surface b.Called theACTIVE SITE c.Only substrates with a shape compatible to the active site can bind 2.Only a small concise area in the peptide chain is actually involved in thecatalytic activity CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 53.Each enzyme catalyzes one reaction, or a limited group of reactions; this isa function of the enzymes SPECIFICITY 4.Four types of enzyme specificity: (lock and key concept) a.Absolute: catalyzes a unique reaction and no other; most enzymesdisplay this type of specificity b.Group: shows broader specificity for a range of substrates withsimilar structures c.Bond: act on certain types of bonds (glycosidic bonds ofcarbohydrates, peptide bonds of proteins) d.Sterioisomeric: react only with certain optical isomers (D and Lisomers of glucose) II.Measurement of Enzymes A.Basics ofenzyme assays 1.Involve kinetic reactions (kinetic = rate); we measure the catalytic ACTIVITY of the enzyme 2.The rate of converting substrate to product is determined a.Amount of product formed per unit of time b.Amount of substrate consumed per unit of time 3.Usually a spectrophotometric measurement when constant reaction rate isreached (ABS/unit of time) B.Endpoint Reaction = single-point, fixed-time 1.Reaction is started and a baseline ABS rdg (A1) is taken; reaction isallowed to incubate at a constanttemp for a fixed period of time(3 min) 2.Reaction is stopped and ABS rdg (A2) is taken to determine amount ofproduct produced in unit of time 3.Assumes a constant amount of product is produced throughout the entireassay period (assumes reaction progress is linear and follows zero orderkinetics) CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 6C.Kinetic Reaction = multiple-point, continuous monitoring 1.The rate of product formation is monitored throughout reaction period at specific time intervals(every 15 sec, 30 sec or 60 sec) 2.Easier to demonstrate approximateLINEARITY of the reaction period 3.Most often used, most practical withmodern instruments/microprocessors,most accurate measurement D.Coupled enzyme reactions are common 1.Involve more than one enzyme 2.Rate-limiting step is initial reaction3.Final reaction is indicating step E.Phases of an enzyme reaction 1.Lag phase: time needed for mixing of reaction components, formation ofE-S complex and reaching thermal equilibrium a.Little or no change ofabsorbance per unit of time b.Occurs virtually immediately 2.Linear phase: amount of product produced per unit of time is constant a.All active sites of enzyme are saturated with substrate b.Product increases linearly with respect to time c.Substrate is in excess (rate of enzyme reaction is independent ofsubstrate concentration = zero order kinetics) d.Measure enzyme activity during this phase (ABS/unit of time) 3.Substrate depletion phase: a.[Substrate] no longer in excess b.Much of the substrate has been converted to product;eventuallyno more product is produced c.Thus, little/no change inabsorbance/unit of time d.Period of reduced velocity:first order kinetics -D-Glucose + O2 + H2Oglucose oxidase gluconic acid + H2O2 H2O2 + reduced chromogenperoxidase oxidized chromogen + H2O CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 7F.Michaelis-Menten Curve 1.Relates velocity of enzyme activity (rate of product formation) to substrateconcentration (a rectangular-hyperbola curve) 2.VELOCITY of enzyme reaction plotted versus SUBSTRATE conc. 3.Increasing [substrate] will increase velocity of reaction a.Rate of reaction is almost directly proportional to [S] b.At low [S], only a fraction of enzyme is associated with substrateas ES complex c.FIRST ORDER kinetics 4.Maximum velocity, Vmax, is reached when substrate is in excess a.At high [S], all the enzyme is bound to substrate as ES complex b.Increasing [S] will not affect reaction rate c.Amount of product formed is proportional to [enzyme] d.ZERO ORDER kinetics 5.Michaelis-Menten curve can be described by mathematical equation: v =Vmax [S] Km+[S] a.Vmax is the maximal rate of reaction when the enzyme is saturated with substrate b.Km , the Michaelis-Menten constant, is the [substrate] that produces one half the maximal velocity (1/2 Vmax) c.Common condition for assaying enzyme activity is a high substrate concentration: [S] ~ 10 x Km , where the rate produced is greater than 90% Vmax ( best if [S] ~ 100 x Km) CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 8G.Lineweaver-Burk Transformation (textbook, page 240-241) 1.Michaelis-Menten curve transformed into a straight line 2.Enables Km and Vmax values to be determined with greater accuracy 3.Advantage: can visualize effect of inhibitors, activators, coupled reactions III.Factors affecting the rate of enzyme-catalyzed reactions A.Temperature 1.Increasing temperature increases the rate of the reaction 2.Doubling of activity for every 10oC (Q10 rule) 3.Certain temperatures produce a specific enzyme effect a.Optimum temp yields maximum activity b.Excessive temps will inactivate the enzyme c.Typically 37o or 30oC are used in enzyme assays B.pH and Buffers 1.Changes in pH will affect the rate of the enzyme-catalyzed reaction 2.pH is controlled by the use of buffers 3.Certain pH will produce a specific enzyme effect a.Optimum pH yields maximum activity pH 4.0 acid phosphatase pH 10.5 alkaline phosphatase b.Extremes in pH (high or low) will inactivate the enzyme c.Changes in pH can change the direction of certain reactions LactateLD, pH 9 Pyruvate PyruvateLD, pH 7Lactate C.Substrate Concentration (Michaelis-Menten theory) 1.Low levels of [substrate] affect formation of the E-S complex a.If fixed [enzyme], rate of product formation is proportional to[substrate] b.First order kinetics c.Rate of reaction dependent on number of active sites filled withsubstrate 2.High levels of [substrate] do not affect formation of E-S complex a.Rate of product formation is not affected by [substrate]b.Maximum rate of reaction (Vmax) is achieved c.Zero order kinetics d.Enzyme concentration affects rate of reaction CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 9D.Enzyme Concentration (Michaelis-Menten theory) 1.Rate of product formation is proportional to enzyme concentration ifsubstrate in excess (zero order kinetics) 2.The higher the enzyme level, the faster the reaction will proceed(because more enzyme is present to bind with substrate) E.Inhibitors 1.Interfere with the reaction, preventing enzyme activity (and product formation); act by a.Removing activators (chelating ions) b.Binding to active site c.Binding to allosteric site (a cavity other than the active site, maybind regulator molecules and be significant to the enzymestructure) 2.Competitive inhibitor a.Binds to active site of free enzyme formingI-E complex b.Competes with substrate for enzyme active sites c.Product does not form from the I-E complex d.Generally reversible by adding more substrate, because thesubstrate is more likely to bind to the active site than the inhibitor 3.Noncompetitive inhibitor a.Binds to regulatory or allosteric site of free enzyme forming I-Ecomplex b.Product does not form from the I-E complex c.Not reversed by adding more substrate, since the inhibitor binds the enzyme independently from the substrate 4.Uncompetitive inhibitor a.Binds to the enzyme-substrate complex forming E-S-I complex b.Product does not form from the E-S-I complex c.At low [S], adding more substrate will cause more inhibition d.Increasing [S] results in more E-S complexes for the inhibitor to bind 5.Each type of inhibition is unique with respects to the effect on the Vmaxand Km of enzymatic reactions and is visualized on the Lineweaver Burk transformation (textbook, page 241) CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 10F.Laboratory Sources of Error 1.Hemolysis: falsely increased LD 2.Lipemia: spectrophotometric analysis 3.Storage conditions: room temp, refrigerator, freezer 4.Glassware contamination: detergents, bleach5.Temperature of reaction 6.Avoid repeated freeze/thaw cycles IV.Enzyme Activity: Units of Measure A.Difficulty in measuring enzyme concentration 1.True serum concentration of most enzymes is minute compared to otherproteins a.Albumin 3.5-5.0 g/dl b.Acid phosphatase in /dl 2.Instability of most enzymes a.Acid phosphatase (pH and temperature) b.Creatine kinase (temperature) B.Measuring enzyme activity 1.Powerful catalytic ability of enzymes employed in analysis a.Rate of reaction is proportional to enzyme concentration in zeroorder kinetics b.Change in ABS/time reflects [product] formed 2.General definition of enzyme activity is the rate of reaction determined bymass of product formed per unit of time in a volume or mass of specimen 3.International Unit of Activity (U) adopted for serum/blood enzymeanalysis under standard conditions: a.The amount of enzyme that will catalyze the conversion of1 micromole of substrate to product per minute, undercontrolled conditions b.Controlled conditions:pH, temperature, substrate, activators c.Most often expressed as unit/volume of specimen (U/L, IU/L) 4.Relating IU to Spectrophotometric Analysis a.A= abc(a = absorptivity coefficient) b.Since the units are similar, IU can be incorporated into the sametype of equation: IU/L=(ABS/min) x (1 cm) x (1000 mole/mmol) x (total vol in mL) (6.22 x 103 mol/L)x(sample volin mL) CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 11V.Coenzymes:NAD NADHorNADH NAD VI.Enzyme Classification A.Six classes based on type of reaction catalyzed 1.Oxidoreductase:catalyze an oxidation-reduction reaction between two substrates (an electron transfer reaction) a.General reaction catalyzed: A (reduced) + B (oxidized)A (oxidized) + B (reduced) b.May involve oxygen: oxidase Example: Glucose oxidase c.May involve hydrogen: dehydrogenase Example: Lactate dehydrogenase (LD) d.May be a reductase or peroxidase 2.Transferase: catalyzes the transfer of a group other than hydrogen fromone substrate to anothera.General reaction catalyzed: A-O+BA+ B-O b.May involve amino acid transfer: transaminase Example: Aspartate transaminase (AST) Alanine transaminase (ALT) Gamma glutamyl transferase (GGT) c.May involve phosphoryl group transfer: kinase Example: Creatine kinase (CK) (requires Mg++) d.May involve carboxyl, glucosyl, methyl groups CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 12 3.Hydrolase: catalyze hydrolysis of various bonds (H2O cleavage of carbonbonds) a.General reaction catalyzed: A-B + H2O A-OH + B-H b.Common enzymes in this category 1)Esterase:cholesterol esterase,pseudocholinesterase 2)Phosphatase: alkaline phosphatase, acid phosphatase 3)Others:amylase, lipase, trypsin, urease, pepsin 4.Lyase (synthetase): catalyze removal of groups from substrates withouthydrolysis.The product contains double bonds (C=N; C=O; C=C) a.General reaction catalyzed A+BA-B (C=C)Synthetase A-B (C=C)A + BLyase b.Example: aldolase, carbonic anhydrase 5.Isomerase:catalyze the interconversion of geometric, optical orpositional isomers (molecular structural rearrangements) a.General reaction catalyzed ABC ACB (same chemical formula) b.Epimerases, mutases or general isomerases Example: phosphohexose isomerase 6.Ligase (synthetase): catalyzes the joining of two substrate molecules,coupled with breaking of the pyrophosphate bond in ATP or a similarcompound (involves phosphorylation from ADP and Pi and bondcleavage) a.General reaction catalyzed A + B + ATP A-B + ADP + Pi (uses energy) A-B + ADP + Pi A + B + ATP (produces energy) b.Example:many enzymes in protein, DNA and RNA synthesis;glutamine synthetase B.Enzyme Nomenclature 1.Each enzyme named by common or trivial names, abbreviationLactate dehydrogenase= LD 2.Enzyme Commission (EC) of the International Union of Biochemistry(IUB) have given systematic names and numbers coding class andsubclasses.Ex: l-lactate:NAD+ oxidoreductase 1.1.1.27 CLS 414 Clinical Chemistry:Student Lab RotationEnzymes Lecture Handout 13VI.Enzymes of Clinical Significance A.Creatine Kinase 1.Specimen collection and handling a.Serum is preferred, especially if fractionation/electrophoresis alsoorderedb.Heparinized plasma may be acceptable;Many anticoagulants (fluoride, EDTA, sodium citrate) inhibit enzyme activity and required activators (Ca2+, Mg2+) c.No hemolysis:intracellular components interfere with chemicalreaction in laboratory measurements (NADH, ATP, HK, etc) d.Avoid lipemia, especially for spectrophotometric analysis e.Relatively unstable: analyze within 4 hours of collection, elsestable at 2-6oC for up to one week; stability increases when storedat lower temps (-20oC) 2.Wide tissue distribution: injury, tissue necrosis will cause elevation of thisenzyme in plasma/serum a.Skeletal muscle b.Heart tissue c.Brain, nerve tissue 3.Physiologic reaction: CK is a transferase: ATP storage and generation incontractile or transport systems, predominantly in muscle cells. Creatine+ATPCKcreatine phosphate+ADP 4.Method of measurement Most common method: modified Rosalki-Oliver method creatine phosphate+ADPCK creatine +ATP ATP + glucoseHKADP +glucose-6-phosphate Glucose-6-phosphate +NADP+G6PD6-phosphogluconate+NADPH (Increased ABS at 340 nm) 5.Isoenzymes: clinical relevance of CK activity often depends more onisoenzyme fractionation than on total levels:a.Isoenzymes occur as a dimer:1)B polypeptide units 2)M polypeptide units b.Three isoenzyme forms: numbered by their anodal (+)electrophoresis mobility with fraction 1 migrating fastest towards the anode 1)CK1=CK-BB (brain type)Normal: ~0% 2)CK2=CK-MB(hybrid, heart)Normal: