Lecture 7 Flashcards

Question

TEMPERATURE

Answer 1

* each enzyme has optimum temp. for max. activity -velocity of reaction increases as temp. increases * there is an optimal temp. at which reaction is most rapid * velocity roughly doubles with a 10C rise in temp. up to about 50C above this temp. reaction rate decreases sharply due to enzyme denaturation by heat * store plasma/serum in fridge or freezer so you dont loose activity of enzyme *temperature must be strictly controlled -30-37 is common and sometimes preincubation is required

Answer 2

* with constant temp. & excess substrate the longer the reaction is allowed to proceed, the more product will be produced *in enzymatic assays, timing is crucial *some enzymes are unstable & start to denature even at RT * when test reaction mixture reaches required temp. the timing of the reaction begins * if the reaction time is too long, the amount of substrate is depleted * reaction time & the analysis time are controlled by the instrument’s test parameters

Answer 3

Act to reduce the reaction rate ▪ decrease or prevent full enzyme activity Small molecules or ions ▪ vary in specificity ▪ single reaction or a range of different ones Reversible ▪ competitive, noncompetitive, allosteric Irreversible ▪ Permanent, removes activities /bioavailability

Answer 4

▪ structurally similar to the enzyme’s substrate (structural analog) ▪ binds to the active site ▪ competes with substrate ▪ forms an enzyme inhibitor (EI) complex ▪ prevents formation of ES complex ▪ Amount of inhibition depends on the relative concentrations of both the substrate & the inhibitor ▪ inhibition can be reversed by increasing [S] ▪ e.g. Hexokinase acts on D-glucose, D-xylose is a competitive inhibitor

Answer 5

▪ structurally different from the substrate ▪ binds to another site ▪ does not compete with substrate ▪ forms an ESI complex ▪ ES formation is Ok but prevents formation of product ▪ addition of more substrate has no effect

Answer 6

▪ also called feedback inhibition ▪ a form of reversible, noncompetitive inhibition ▪ product binds to a regulatory site on an enzyme located upstream ▪ feature of enzymes involved in metabolic pathways ▪ mechanism for controlling the rate of synthesis of a metabolic intermediate according to need ▪ when level of required chemical compound drops below its required concentration, the enzyme which catalyzes the production becomes unlocked and reaction proceeds again

Answer 7

▪ Inhibitor binds covalently ▪ Progressive with time ▪ eventually becomes complete ▪ Increased [S] will not reverse inhibition ▪ Examples: ▪ nerve gases and pesticides ▪ cyanide blocks cellular respiration

Answer 8

relationship between velocity of an enzymatic reaction and substrate concentration X axis * substrate concentration * [S] is variable Y axis * velocity of the reaction rate * expressed as amount of products formed per unit of time * [E] is fixed (in specimen) Vmax * point at which the reaction is proceeding at its maximum velocity (maximum rate of catalysis) * point at which reaction rate is independent of substrate conc’n * all enzyme is in E-S complex ½ Vmax * point where reaction proceeds at half its maximum velocity * half of the enzyme is in E-S complex Km (Michaelis-Menten constant) *substrate concentration S at which reaction proceeds at half its maximum velocity (½ Vmax) * expresses affinity of substrate & enzyme * increased affinity gives a lower Km * substrate concentration used for measuring enzyme activity is 20-100 x Km ensures that enzyme will be in E-S

Answer 9

* use zero-order reaction kinetics * substrate is present in excess of enzyme (ie. there is enough to bind all enzyme in specimen) * reaction rate is independent of [S] * reaction rate depends only on [E] in specimen * if we add more substrate, there will be no further increase in reaction rate * this type of reaction used to measure activity (amount) of an enzyme in a patient specimen

Answer 10

* [E] is fixed * [S] is variable * enzyme is present in excess of substrate * constant amount of enzyme (in reagent) reacts with increasing concentration of substrate (in specimen) * reaction rate steadily increases as more substrate added * reaction rate is dependent & directly proportional to [S] * this type of reaction used when an enzyme is used as a reagent in order to measure another analyte

Answer 11

* most enzymes are in very low concentration in serum or plasma * lab assays measure catalytic activity not concentration of enzymes in specimens * activity measured is dependent on enzyme concentration * rate of an enzyme-catalyzed reaction is directly proportional to amount of active enzyme in a specimen * elevation of enzyme activity is indicative of some pathological process Methods for measureing enzyme activity 1. measure depletion of a substrate (or coenzyme) *measure the substrate concentration before & after the enzyme reaction occurs * measure a decrease in Absorbance 2. measure the formation of a product * measure after enzyme has acted on the substrate * measure an increase in Absorbance

Answer 12

Fixed Time Methods *reactants are combined * reaction proceeds for a designated time or is stopped * amount of absorbance change produced by enzyme is measured photometrically * the larger the reaction, the more enzyme is present * sometimes called end-point methods * can measure either product formation or substrate depletion * zero time = enzyme & substrate mixed together * incubation time = reaction mixture incubates at a constant temp. for a fixed period * measurement time = at end of fixed time, a photometric reading taken (Absorbance)m measure Absorbance of product formed or substrate depleted at a specific wavelength - the larger the reaction, the more enzyme that is present Assumption: * reaction rate is constant (linear) throughout the entire reaction period * not always true- there is usually an initial lag phase and substrate depletion may occur – timing is important! SLIDE - EXAMPLE

Answer 13

* lag phase: early phase of assay, reactants are mixed & kinetic equilibrium is still being established * substrate depletion :not enough substrate in reagent to bind all of the enzyme in specimen. This occurs when enzyme level is elevated * long assay times can cause enzyme degradation- results in loss of enzyme activity *Upper limit of usefulness may be too low * Unable to shorten incubation period * Dilution may not give a proportionate change in activity * Amount of change measured during fixed-time interval may not reflect zero-order kinetics only measurements taken during the linear phase will provide an accurate result

Answer 14

* progression of enzymatic reaction is continuously monitored as it occurs * multiple absorbance readings are taken at specific time intervals or continuously by a continuous recording spectrophotometer in a cuvette starting at zero time over a defined period * also called * multi-point methods * kinetic methods * rate methods * measure change in Abs. per unit of time the formula is A/T *change in Absorbance per unit of time is proportional tothe enzyme’s activity in specimen

Answer 15

* earliest time in an enzymatic assay where reactants (enzyme & substrate) are first mixed together * reactants are reaching the defined temp. & kinetic equilibrium * Do not take Absorbance measurements yet Linear Phase * there is a linear change in absorbance over time *[S] is in excess of enzyme * rate of reaction is independent of [S] * assay follows zero order kinetics * slope = reaction rate = A/T SUBSTRATE DEPLETION PHASE * occurs late in reaction when [S] falls * insufficient substrate to bind all enzyme in specimen * specimen contains very high levels of an enzyme * reaction no longer follows zero order kinetics - most instruments give warning * dilute specimen with water or saline & re-analyze. Dilution reduces amount of enzyme in reaction mixture SEE SLIDE

Answer 16

* reaction time can be shorter than fixed time * instrument can delay readings until lag phase over * instruments capable of making hundreds of readings over a short period of time * identifies substrate depletion (due to high enzyme activity) * upper limit of enzyme activity that can be measured is higher than fixed time * The preferred method!

Answer 17

*catalytic activity, we need activity units * unit (IU) defined as amount of enzyme which catalyzes 1 micromole (umole) of substrate per minute per litre (L) of serum *each lab has its own intervals SEE EXAMPLE SLIDE

Answer 18

* enzymes also used in reagents to measure other analytes in specimens using first-order reaction kinetics * serum/plasma contains the analyte to be measured which is the enzyme’s substrate → [E] is fixed and in excess * reagent contains the enzyme which will catalyze a specific reaction involving the analyte to be measured→[S] is variable * eg. Glucose assay * uses an enzyme, hexokinase * converts glucose +ATP to Glucose 6 Phos +ADP which acts in a second reaction with coenzyme NAD & can be measured photometrically SEE REACTION EXAMPLE

Answer 19

Plasma-specific enzymes * thrombin, Factor XII, Factor X, plasminogen (coagulation) Secreted enzymes * salivary glands, pancreas (lipase, amylase, trypsinogen) * prostate (prostatic acid phosphatase, prostate-specific antigen) Cellular enzymes * Lactate dehydrogenase (LD) * aminotransferases (AST, ALT) * alkaline phosphatase (ALP

Answer 20

* Intracellular enzyme Elevated in: * myocardial infarction (LD1,2) * hemolysis * megaloblastic anemia (LD1,2) * artefactual hemolysis * pulmonary embolism (LD3) * liver disease (LD4,5) * differentiate using isoenzyme patterns

Answer 21

* Intracellular enzyme Elevated in: * hepatobiliary disorder * biliary tract obstruction induces ALP synthesis * bone disorders * Paget’s disease * fractures * normal growth, pregnancy

Answer 22

* leakage from cells due to cell damage or cell death (necrosis) * cell membrane deterioration due to microbiological agents, organic chemicals -causes decreased tissue perfusion leading to myocardial infarction, liver hypoxia, shock -effects skeletal muscle Altered production *Induction * alkaline phosphatase and normal bone growth, bone disease, pregnancy, biliary obstruction - GGT, drugs Reduced clearance *urinary excretion is not a major route for elimination as these proteins are generally too large to pass through the glomerulus