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Lecture 7 Flashcards

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1
Q

what are enzymes, what is there use and where are they found

nomenclature

A
  • proteins that catalyze biochemical reactions
  • equilibrium point not altered
  • enzyme is not consumed or changed in composition
  • substrates converted to products will not happen without specific enzymes
  • increase the rate of reactions as much as 107 fold
    *activity of enzymes can be measured to determine the location and nature of pathological changes in the tissues and organs in the body
  • produced intracellularly and function in the cell
    -found in all body tissues
    -some just in plasma
  • can appear in serum following cellular injury or from degraded cells

creatine kinase (MI), amylase (acute pancreatitis)
like pepsin - breaks proteins into peptides

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2
Q

what are some properties of enzymes and what shapes are they in

A

Enzymes are proteins so they have
* Primary structure: line of amino acids linked by peptide bonds
* Secondary structure: alpha helix, beta pleated sheets
* Tertiary structure: 3D structure (binding and folding)
* Quaternary structure: polypeptides or dimers (2 polypeptides) creatine kinase (CK) found in muscle or *lactate dehydrogenase (LD) found in muscle, liver, red blood cells

-can be ionized in solution : cation or anion depending on pH
-can be simple proteins like pepsin or trypsin or they can be conjugated proteins

-can be denatured by heat, organic solvents, heavy metals and pH causing inactivation

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3
Q

Enzyme structure

A
  • each enzyme has an active site
    ▪ a water free cavity where the substrate interacts with charged amino acid residues of the enzyme
  • enzymes have an allosteric site
    -a cavity other than an active site
    -may bind regulatory molecules
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4
Q

Holoenzyme = * Apoenzyme +* Cofactor

A
  • Holoenzyme: complete & active enzyme system * (protein portion + non-protein portion)
  • Apoenzyme * protein portion (polypeptide) * large molecule * less active or inactive without a cofactor
  • Cofactor * non-protein molecule, binds the enzyme before the reaction will occur
  • enhances or activates activity of apoenzyme
    • coenzyme (organic, loosely bound),
    • prosthetic group (organic, tightly bound)
      - metal ion (inorganic)
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5
Q

types of cofactors

A

Coenzyme:
* organic compound
* loosely or only momentarily attached to the apoenzyme
* carrier of an electron, atom or functional group
* often a vitamin (B vitamin)
* co-substrates
* Carrier for electrons, phosphate groups
* E.g. NAD, NADP

Prosthetic group:
* permanently bound to apoenzyme
* forms part of the active centre
* undergoes chemical change during reaction
* E.g heme

Activators
* metal ions
* inorganic
* divalent cations: Mg2+, Fe2+, Zn2+; sometimes anions: Cl-
* structural stability
* play a direct role in catalysis
* firmly or weakly incorporated
1. metalloenzyme
* metal ion is an essential structural
component of enzyme
* E.g. zinc in carbonic anhydrase
2. metal-activated enzyme
* metal ion must be present for full
activity
* E.g. Mg++ must be present for DNAse,
RNAse to be activated

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6
Q

Another form of enzyme activation * Proenzymes (zymogens)

A
  • structurally inactive enzyme or precursor
  • manufactured & secreted by cell in an inactive form
  • converted into active enzyme by an activator
  • like digestive enzymes in that it protects tissue from auto digestion

pepsinogen + H+ in to pepsin

trypsinogen + enterokinase into trypsin

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7
Q

Isoenzymes

A
  • Enzymes can exist in different forms, called Isoenzymes (Isoforms)
  • functionally identical – same chemical activity
  • physically different – identical active centers, but different arrangement of amino acids on side chains.
  • mixtures of different subunits with different genes, produced in different tissues
  • example:
  • Creatine kinase (CK) 3 isoE
    - CK1 (BB) * brain, bladder, GI tract
    - CK2 (MB) * cardiac muscle *
    - CK3 (MM) * skeletal muscle *
  • Lactate dehydrogenase (LD) 5 isoE
    - LD1 & 2 * cardiac muscle
    * LD4 & 5 * specific to liver

-measuring isoenzymes allows us to determine which specific tissue is damaged
* mobility on electrophoresis
* resistance to heat denaturation
* solubility
* catalytic characteristics:
* speed of reactions with different substrates
* response to inhibitors

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8
Q

What is a catalyst?

A
  • substance that increases the rate of a chemical reaction by decreasing the activation energy required
  • substance is not consumed or permanently altered
  • does not alter the equilibrium of reaction
  • inorganic compounds
  • can perform at extreme temperatures & pH
  • non-specific
  • enzymes are protein catalysts of biological origin
  • Organic compounds
  • perform at physiological temperature & pH
  • specific for certain reactions- determined by 3ary structure
  • more efficient than inorganic catalysts
  • easy to detect its presence

LOOK AT SLIDE

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9
Q

INDUCED FIT

A
  • enzyme undergoes a change in conformation when it reacts with a substrate to form E-S complex
    -site held by weak H and ionic bonds
  • substrate induces a structural change in
    enzyme
  • active site consist of 2 components
    * one for substrate specificity
    * one for catalysis
  • active site is flexible, it can be induced to fit several structurally similar compounds but willb only be active if there is correct alignment of the substrate
    -active site and R groups of its aa can lower activation energy and speed up the rate of reaction by acting as a template for substrate orientation, stressing the substrate and stabilizing the transition state, give a favorable micro environment

HEXOKINASE AND GLUCOSE

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10
Q

Enzyme Specificity

A
  • the specificity of an enzyme is its ability to catalyze one (or more) specific reactions
  • is one of its most important properties
  • composition & spatial arrangement of active center is basis for its specificity

Absolute (substrate) specificity
* only one substrate, only one reaction
* glucose but not lactose or any other sugar

Bond specificity
* peptide bonds between two amino acids
* ester linkages in lipids

Group specificity
* substrates with a particular chemical group
* phosphate esters

Stereoisomeric specificity
* D-glucose not L-glucose, Beta but not alpha

  • Uricase : substrate specific – acts only on uric acid
  • Urease: substrate-specific: acts only on urea
  • Lipase: bond-specific: ester linkages in lipids
  • Pepsin, trypsin: bond-specific: peptide linkages in proteins
    *Phosphatase :group-specific: cleave phosphate groups * alkaline phosphatase, acid phosphatase
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11
Q

enzymes in disease

A
  • enhanced leakage of intracellular contents (including enzymes found in the cell) is an indication of disease
  • enzyme specificity gives us info on pathology
  • Examples
  • gamma -glutamyl transferase (GGT) in alcoholic cirrhosis
  • amylase, lipase in pancreatitis
  • creatine kinase in myocardial infarction
  • aspartate aminotransferase in liver disease
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12
Q

NAMING

A

enzyme acts + (ase)
* urease acts on urea
* amylase acts on amylum (starch)
* systematic name, based on naming and classifying of enzymes by type of chemical reaction & reaction mechanism
-each enzyme has an EC number -Enzyme Commission - 4 DIGITS

  • 1st digit: places enzyme in one of six classes
  • indicates type of reaction catalyzed
  • 2nd digit: subclass
  • indicates group transferred
  • 3rd digit: sub-subclass
  • indicates group accepted
  • 4 th digit: specific serial number
  • assigned in the order enzyme was isolated & classified
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13
Q

CLASS 1 - Oxidoreductase

A

Lactate + NAD <-LD-> pyruvate + NADH

Ared + Box <—-> Aox + Bred

  • catalyze an oxidation-reduction reaction (electron transfer) between 2 substrates
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14
Q

CLASS TWO Transferase

A

L-glutamate + oxaloacetate <-AST-> L-aspartate + alpha- ketoglutarate

A-X + B <–> A + B-X

  • catalyze transfer of a chemical group from one substrate to another
  • a new amino acid and a new keto acid are formed
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15
Q

CLASS 3 . Hydrolase

A

starch + H2O –AMYLASE–> maltose + dextrins

triglyceride + H2O—-LIPASE-> glycerol + fatty acids

  • catalyze breakdown of bonds with the addition of a water molecule (hydrolysis)
  • some enzymes in this class associated with digestion (breakdown of CHO, proteins, lipids)
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16
Q

CLASS 4 Lyase

A

Carbonic Anhydrase
H2CO3 <—CA—–> CO2 + H2O

Aldolase
Glucose —– ALDOLASE——> 2 Trioses

*catalyze removal of groups from substrates without hydrolysis
* the product contains double bonds
* or it may catalyze the reverse reaction by adding a group to a double bond

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17
Q

CLASS 5 Isomerase

A

glucose-6-phosphate TO fructose-6-phosphate WITH ketol isomerase

ALPHA -D-glucose TO BETA -D-glucose WITH mutarotase

  • catalyze change of one geometric or optical isomer into another
  • structural or geometrical changes are made within a molecule (internal rearrangement of atoms)
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18
Q

CLASS 6 Ligase

A

also called synthetases
* A + B + ATP <——–> AB + ADP + Pi
* DNA fragment 1 + DNA fragment = 2 DNA strand
* catalyze joining of 2 substrate molecules, coupled with the breaking of a bond in adenosine triphosphate (ATP) or a similar
triphosphate

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19
Q

FACTORS GOVERNING ENZYMATIC REACTIONS

A

▪ substrate concentration
▪ enzyme concentration
▪ pH
▪ temperature

MODULATORS
▪ activators
▪ inhibitors

20
Q

SUBSTRATE CONCENTRATION

A

-as constant enzyme concentration increases so does the rate (velocity) of enzymatic reaction until a max rate is reached
-once this rate is reached you can add more enzyme but there will be no increase in rate of reaction - ENZYME SATURATED all in the complex

Low Substrate Concentration
▪ concentration is rate limiting- not enough to keep the reaction going
▪ enzyme cannot work to its maximum capacity

As Substrate Concentration Increases
▪reaction rate increases proportionally with substrate concentration
▪ reaction rate is directly proportional to substrate concentration

  • At Maximum Substrate Concentration
    ▪ all enzyme is working at maximum capacity
    ▪ all enzyme is in E-S complex
    ▪ reaction velocity has reach maximum - Vmax
    ▪ enzyme activity and reaction rate is independent of substrate concentration

in lab substrate concentration of 20-100
times greater than the required concentration so that enzyme is all in E-S form

21
Q

FIRST ORDER KINETICS

A
  • if enzyme concentration [E] is fixed, then for first-order kinetics
    ▪ reaction rate is directly proportional to substrate concentration [S] at low values
    ▪ the enzyme does not have enough work to do
    ▪ the formation of product will proceed faster if you add more substrate; the reaction rate will increase in proportion to the amount of substrate added as well
22
Q

ZERO ORDER KINETICS

A
  • if enzyme concentration [E] is fixed, then for
    zero-order kinetics
  • reaction rate is independent of [S] at high values
  • reaction velocity is at maximum
  • enzyme is “saturated”
  • amount of substrate added will eventually exceed the amount of enzyme present
  • enzyme now be working at maximum capability
  • as soon as it releases product, it will combine with new substrate
  • adding even more substrate: no effect on reaction rate therefore independent of [S]
    * now zero-order kinetics
23
Q

ENZYME CONCENTRATION

A

▪enzyme concentration is measured by its catalytic activity
▪ when substrate concentration is in excess, reaction velocity is proportional to enzyme concentration
▪ more ES complexes formed

Basis for the quantitative determination of enzymes:
▪ measure amount of product formed
- if time is kept constant, products are doubled with a 2-fold increase in enzyme
-reaction rate has doubled

▪ measure amount of substrate used
-if time is kept constant, substrate is depleted twice as quickly with a 2- fold increase in enzyme
* reaction rate has doubled

24
Q

PH

A
  • enzymes (as proteins) are sensitive to pH
  • each has optimum pH for maximum activity
  • extreme changes in pH - denaturation
  • small changes in pH - * alter degree of ionization or interfere with ES complex formation or loss of catalytic activity
  • pH for a forward reaction may be different than the pH for the reverse reaction (LD)
    -in lab it is essential to keep pH constant to measure activity
25
Q

TEMPERATURE

A
  • 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
26
Q

TIME

A
  • 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

27
Q

INHIBITORS

A

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

28
Q

COMPETITIVE INHIBITOR

A

▪ 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

29
Q

NONCOMPETITIVE INHIBITOR

A

▪ 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

30
Q

ALLOSTERIC INHIBITOR

A

▪ 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

31
Q

iRREVERSIBLE INHIBITION

A

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

32
Q

MICHAELIS-MENTEN CURVE

A

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
33
Q

MEASUREMENT OF ENZYME ACTIVITY

A
  • 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
34
Q

FIRST ORDER REACTIONS

A
  • [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
35
Q

QUANTITATIVE ENZYME
MEASUREMENT

A
  • 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

  1. measure the formation of a product
    * measure after enzyme has acted on the substrate
    * measure an increase in Absorbance
36
Q

HOW DO WE MEASURE THE EXTENT
OF AN ENZYMATIC REACTION? Fixed Time Methods

A

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

37
Q

FIXED TIME METHODS - DISADVANTAGES

A
  • 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

38
Q

HOW DO WE MEASURE THE EXTENT
OF AN ENZYMATIC REACTION?
CONTINUOUS-MONITORING METHOD

A
  • 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
39
Q

Lag Phase and Linear Phase, SUBSTRATE DEPLETION PHASE in CONTINUOUS-MONITORING

A
  • 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

40
Q

CONTINUOUSLY MONITORED METHOD
* Advantages

A
  • 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!
41
Q

UNITS OF ENZYME ACTIVITY

A

*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

42
Q

ENZYMES AS REAGENTS

A
  • 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

43
Q

CLASSIFICATION OF ENZYMES IN BLOOD

A

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

44
Q

ENZYMES OF CLINICAL SIGNIFICANCE Lactate dehydrogenase (LD)

A
  • 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
45
Q

ENZYMES OF CLINICAL SIGNIFICANCE
* Alkaline phosphatase (ALP)

A
  • Intracellular enzyme
    Elevated in:
  • hepatobiliary disorder
    • biliary tract obstruction induces ALP
      synthesis
  • bone disorders
    * Paget’s disease
    * fractures
  • normal growth, pregnancy
46
Q

ENTRY OF ENZYMES IN BLOOD

A
  • 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

Chemistry (12 decks)

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