C1 - Section 1. INTRODUCTION TO ENZYMES Flashcards
is a field of laboratory medicine which focuses on the study of enzymes and their significance to the diagnosis and treatment of diseases.
Clinical Enzymology
These are substances that catalyzes a given chemical reaction
enzymes?
The reaction they catalyze are frequently (?) which means which means that they can synthesize and decompose molecules
reversible
They are (?) types of protein in terms of both structure and function
complicated
They easily (?) with varying molecular weight and mass
denatured
These enzymes are (?) which are capable of ionizing either as acid or base
amphoteric
They are synthesized in an (?) and operates in the presence of a (?)
inactive state
cofactor
Enzymes are found in all body tissues, they appear in the serum following cellular injury or they may come from degraded cells thus changes in (?) reflects changes in state of health.
enzyme concentration
non-protein organic biochemical that takes part in the enzyme reaction
Coenzymes
Essential to the catalytic activity as a CO-SUBSTRATE
Coenzymes
Diffusible, heat stable, low molecular weight that when combined tightly to enzymes, the coenzyme will be called Prosthetic group
Coenzymes
Coenzymes E.g.
NAD, Pyridoxal phosphate
- Inorganic ionic cofactor
Activators
increase the catalytic activity of an enzyme when it binds to specific site
Activators
Metabolic regulator of enzyme reaction
Activators
Usually metal ions (esp. divalent cations)
Activators
Activators E.g.
Mg++, Na+, K+, Zn++
- the combined enzyme & coenzyme
Holoenzyme
- Enzyme without a cofactor
Apoenzyme
- A coenzyme that cannot be removed from its attachment to an enzyme using dialysis
Prosthetic Group
Prosthetic Group E.g.
Pyridoxal phosphate in transaminase reaction
- Substance acted upon by an enzyme & is converted into a new substance
Substrate
- Substance derived from a transformed substrate
Product
– Site where substrate interacts with enzymes
Active site
– Site other than the active site that may lead to either attachment of substrate to the enzyme’s active site or inhibition of attachment
Allosteric site
– different form of an enzyme with different genetic origins but catalyze the same reaction
Isoenzymes
– Results when an enzyme is subject to different post-transitional modification
Isoforms
- Refers to the sequence of amino acids joined by peptide bonds to form a polypeptide chain
a. Primary Structure
- Conformation of the segments of polypeptide chain
b. Secondary Structure
Made up of alpha helices or beta-pleated sheets which are maintained by hydrogen bonds
b. Secondary Structure
- Arises from the interactions among side chains/groups of the polypeptide chain
c. Tertiary Structure
Structure are bent and folded and maintained by covalent disulfide bond
c. Tertiary Structure
- Separate bended & folded structures are put together to form a functional unit
d. Quarternary Structure
Enzyme variants –
LDH, Creatine kinase
The enzyme action model
a. Enzymes act through formation of (?)
b. The substrate must be bound to the (?)
c. The (?) will then break down to give the reaction products and free the enzyme
d. All enzyme reactions are in theory (?) however, in practice, reactions are usually more rapid in one direction than the other.
enzyme substrate complex
active site of the enzyme
enzyme-substrate complex
reversible
refers to the active site being complementary in shape & size to the substrate
First presented by Emil Fisher, the lock represents an enzyme and the key represents a substrate.
Lock and Key Theory
It is assumed that both the enzyme and substrate have fixed conformations that lead to an easy fit.
Lock and Key Theory
Because the enzyme and the substrate are at a close distance with weak attraction, the substrate must need a matching shape and fit to join together.
Lock and Key Theory
At the active sites, the enzyme has a specific geometric shape and orientation that a complementary substrate fits into perfectly.
Lock and Key Theory
Factors that influence the enzymatic reaction
– The rate of enzymatic reaction
Time
If the catalytic activity of an enzyme on a substrate is fast, this will mean a shorter reaction time thus liberating the enzyme to act again on the remaining substrate
Time
– Commonness between the enzyme and the substrate
Molecular compatibility
– Number of substrate that can be reacted
Space availability
- capacity of enzymes to recognize and bind only one or few molecules among others
Specificity
– when an enzyme can act and catalyze one unique reaction
Absolute specificity
– when some enzymes act on different substrates belonging to the same group
Group specificity
– an enzyme acts only on the specific isomer
Stereoisomeric
o Enzyme conc. is fixed; Substrate conc. is varied
First order Kinetics
o Rate of reaction is almost directly proportional to substrate conc. at low values
First order Kinetics
o At low concentration of the substrate, only a fraction of the enzyme is associated with the substrate
First order Kinetics
o The rate observed reflects the low concentration of the ES complex
First order Kinetics
o When maximum velocity is reached, the rate of increase in velocity is “O”
Zero order Kinetics
o Reaction rate is unaffected by increased substrate concentration
Zero order Kinetics
o Dependent on enzyme concentration
Zero order Kinetics
o In this reaction, the entire enzyme is bound to substrate and a much higher rate of reaction is obtained
Zero order Kinetics
o Because the entire enzyme is present in the form of the complex, there is now no further increase in ES complex conc. No further increment in reaction rate are possible
Zero order Kinetics
- shows the relationship of the reaction velocity to the substrate concentration
Michaelis-Menten Curve
- °T considered favorable for enzyme activity (30-37°C or 37 – 40°C)
Optimum temperature
– reaction rate is doubled for every 10°C increase
Q10 value
: enzyme undergoes inactivation and denaturation
50 – 60°C
- the point at w/c the reaction rate is greatest
Optimum pH
, many enzymes show maximum activity
At pH 7.0 – 8.0
pH value are seen as
low as 1.5 and as high as 10.5
may be different in forward and reverse reaction
Optimal pH
is important: it affects the three dimensional confirmation of the enzyme
Maintaining pH
– increased reaction rate
Activators
Bind the substrate to the active site by forming ionic bridges
Activators
Orients the substrate so it is attached to the enzyme in the correct configuration
Activators
- Decrease the rate of enzyme reaction
Inhibitors
- Inhibitors Binds to the active site, blocks access of the S to the E
Competitive inhibition
is a structural analog of the substrate, but it is not identical thus breakdown to products do not take place o
competitive inhibitor
If substrate conc. Is significantly higher than the inhibitor, the inhibition may be reversible
Competitive inhibition
- Binds elsewhere on the E causing change in shape that interferes w/ S binding
Non-competitive inhibition
Attachment of the inhibitor to the enzyme does not alter the affinity but the presence of ESI prohibit the formation of products
Non-competitive inhibition
- Inhibition-inhibitors binds to the ES complex, if substrate will be increased, there will be increase in ES conc. increasing inhibition, this inhibition does not yield product.
Uncompetitive inhibition
- Inhibitors are possible removed from the system; enzyme is fully restored
Reversible inhibition
- Inhibitors covalently combine w/ the enzyme
Irreversible inhibition
o Physical methods are ineffective in separating inhibitors from the enzymes
Irreversible inhibition
o Physical processes that remove inhibitors: dialysis, gel filtration
Reversible inhibition
Methods of Enzyme Assay
The reactants are combined
Fixed Timed Assays
Reaction proceeds for a designated time & is stopped (by inactivating the enzyme)
Fixed Timed Assays
reaction is stopped by a weak acid, then measurement is made of the amount of reaction that has occurred
Fixed Timed Assays
The reaction is assumed to be linear over the reaction time, the larger the reaction, the more enzyme is present
Fixed Timed Assays
Multiple measurements are made at specific time intervals or by a continuous-recording spectrophotometer
Continuous-monitoring or kinetic assay
Advantage: Linearity of the reaction is adequately verified
Continuous-monitoring or kinetic assay
This is preferred because any deviation in linearity is readily observable
Continuous-monitoring or kinetic assay
Proposed by the Commission on Enzymes (IUB)
IU – International Unit
Used to standardize the system or reporting of quantitative results
IU – International Unit
is the amount of enzyme that will catalyze the reaction of 1 µmol of substrate per minute under specified conditions of temperature, pH, substrate and activators.
IU – International Unit
Expressed in terms of U/L or mU/L
IU – International Unit
unit of enzyme activity w/c converts 1 mol of substrate per second
Katal
conforms w/ the Systemè International (SI) scheme of units
Katal
Mole is the unit for substrate concentration while the unit of time is second
Katal
Enzyme concentration is then expressed as
katals per liter
Factors that influence rate of entry
Impaired energy production: promote deterioration of cell membrane
Leakage of enzymes from cells
Direct attack on the cell membranes (viruses or organic chemicals)
Leakage of enzymes from cells
Reduction in the supply of oxygenated blood perfusing any tissue (e.g. MI)
Leakage of enzymes from cells
Genetic deficiency of enzyme production
Altered enzyme production decrease
Enzyme production is depressed as a result of disease
Altered enzyme production decrease
– NOT a major route for elimination
Urinary excretion
Except: Amylase = ↑blood levels (e.g. Acute pancreatitis)
Urinary excretion
Inactivated enzymes are removed by the RES
Enzyme Inactivation in Plasma
Half – life in plasma: 24 – 48 hours
Enzyme Inactivation in Plasma
the enzyme changes in shape during binding to accommodate the substrate
Induced fit Model
The induced-fit model is generally considered the more correct version
Induced fit Model
This theory maintains that the active site and the substrate are, initially, not perfect matches for each other.
Induced fit Model
inorganic activators existing as part of the enzyme molecules
Metalloenzyme
Examples of Inhibitors
- Excess substrate
- Product of reaction
- E-S complex does not break to yield products
- Chemical substances
- causes competition between substrate molecules for a single binding site
- Excess substrate
-may be an inhibitor of the forward reaction
- Product of reaction
Disruption of the 3-dimensional structure of the enzyme molecule
ENZYME DENATURATION
ENZYME DENATURATION
• May be reversed if:
•denaturation is not extensive
•denaturing agent is removed
DENATURING CONDITIONS
- Elevated temperature
- Extremes in pH
- Radiation
- Frothing
- Strong salt solution
- Mechanical trauma
- Chemicals
The (?) is directly proportional to the (?) present in the system
rate of an enzyme-catalyzed reaction
amount of active enzyme
SOURCES OF ERRORS IN ENZYME ASSAY
- Use of Plasma
- Hemolysis
- Turbid/Lactassent Serum (Lipemia)
- Heat labile enzyme
- Contaminants
- Least preferred specimen
- inhibitory effects of anticoagulants on enzyme activity
Plasma
Release of intracellular enzyme
Hemolysis
- Inhibits CK & Amylase
Turbid/Lactassent Serum (Lipemia)
