Lecture 7 Flashcards
what are enzymes, what is there use and where are they found
nomenclature
- 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
what are some properties of enzymes and what shapes are they in
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
Enzyme structure
- 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
Holoenzyme = * Apoenzyme +* Cofactor
- 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)
types of cofactors
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
Another form of enzyme activation * Proenzymes (zymogens)
- 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
Isoenzymes
- 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
What is a catalyst?
- 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
INDUCED FIT
- 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
Enzyme Specificity
- 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
enzymes in disease
- 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
NAMING
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
CLASS 1 - Oxidoreductase
Lactate + NAD <-LD-> pyruvate + NADH
Ared + Box <—-> Aox + Bred
- catalyze an oxidation-reduction reaction (electron transfer) between 2 substrates
CLASS TWO Transferase
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
CLASS 3 . Hydrolase
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)
CLASS 4 Lyase
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
CLASS 5 Isomerase
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)
CLASS 6 Ligase
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
FACTORS GOVERNING ENZYMATIC REACTIONS
▪ substrate concentration
▪ enzyme concentration
▪ pH
▪ temperature
MODULATORS
▪ activators
▪ inhibitors
SUBSTRATE CONCENTRATION
-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
FIRST ORDER KINETICS
- 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
ZERO ORDER KINETICS
- 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
ENZYME CONCENTRATION
▪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
PH
- 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
TEMPERATURE
- 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
TIME
- 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
INHIBITORS
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
COMPETITIVE INHIBITOR
▪ 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
NONCOMPETITIVE INHIBITOR
▪ 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
ALLOSTERIC INHIBITOR
▪ 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
iRREVERSIBLE INHIBITION
▪ Inhibitor binds covalently
▪ Progressive with time
▪ eventually becomes complete
▪ Increased [S] will not reverse inhibition
▪ Examples:
▪ nerve gases and pesticides
▪ cyanide blocks cellular respiration
MICHAELIS-MENTEN CURVE
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
MEASUREMENT OF ENZYME ACTIVITY
- 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
FIRST ORDER REACTIONS
- [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
QUANTITATIVE ENZYME
MEASUREMENT
- 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
- measure the formation of a product
* measure after enzyme has acted on the substrate
* measure an increase in Absorbance
HOW DO WE MEASURE THE EXTENT
OF AN ENZYMATIC REACTION? Fixed Time Methods
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
FIXED TIME METHODS - DISADVANTAGES
- 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
HOW DO WE MEASURE THE EXTENT
OF AN ENZYMATIC REACTION?
CONTINUOUS-MONITORING METHOD
- 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
Lag Phase and Linear Phase, SUBSTRATE DEPLETION PHASE in CONTINUOUS-MONITORING
- 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
CONTINUOUSLY MONITORED METHOD
* Advantages
- 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!
UNITS OF ENZYME ACTIVITY
*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
ENZYMES AS REAGENTS
- 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
CLASSIFICATION OF ENZYMES IN BLOOD
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
ENZYMES OF CLINICAL SIGNIFICANCE Lactate dehydrogenase (LD)
- 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
ENZYMES OF CLINICAL SIGNIFICANCE
* Alkaline phosphatase (ALP)
- Intracellular enzyme
Elevated in: - hepatobiliary disorder
- biliary tract obstruction induces ALP
synthesis
- biliary tract obstruction induces ALP
- bone disorders
* Paget’s disease
* fractures - normal growth, pregnancy
ENTRY OF ENZYMES IN BLOOD
- 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
