Enzymes 1 Flashcards
enzymes
specific biologic proteins that catalyze biochemical reactions
- lower the activation energy needed for the reaction to proceed
- denatured by heat, changes in pH, etc
not consumed or changed in composition
Can appear in all body tissues and frequently appear in serum following cellular injury or degradation
may exist in different forms – isoenzymes and isoforms
Enzyme nomenclature
Enzyme Commission (EC) developed a classification system in 1961
• Systematic Name – defines substrates acted on, reaction catalyzed & name of any coenzymes
• Recommended Name – more usable/practical
• Abbreviation – widely used in laboratories
• Code Number – four digit number separated by decimal points
- 1st digit places enzymes into one of six classes
Classes of enzymes
The International Union of Biochemistry (IUB) system assigns a name and code to each enzyme.
- Oxidoreuctases: catalyze an oxidation-reduction reaction between 2 substrates.
- Transferases: catalyze transfer of a group other than hydrogen from one substrate to another.
- Hydrolases: catalyze hydrolysis of various bonds. (H2O added/substrate broken down)
- Lyases: catalyze removal of groups from substrates without hydrolysis; product will contain
double bonds - Isomerases: catalyze interconversion of geometric, optical, or positional isomers- ex. Convert D to L forms
- Ligases: catalyze joining of two substrate molecules coupled with breaking of pyrophosphate bond in ATP
Enzymes we need to know this semester
- LDH – Lactate dehydrogenase
- AST – Aspartate aminotransferase
- ALT – Alanine aminotransferase
- CK – Creatine kinase
- GGT – γ-Glutamyltransferase
- ALP – Alkaline phosphatase
- ACP - Acid phosphatase
- AMS - Amylase
Enzyme specificity
◦ Absolute – specific to only one substrate
ex. urease only catalyzes hydrolysis of urea
◦ Group – specific to all substrates of a chemical group
ex. ALP on phosphate ester group
◦ Bond (linkage) – specific to chemical bonds
ex. trypsin & pepsin act on peptide bonds
◦ Stereoisomeric – specific to one optical isomer of a compound
ex. LD acts on L-Lactate but not D-Lactate
methods to measure enzymes
• Enzyme concentrations in blood are normally very low.
◦ Therefore, increased levels in blood indicate a disease process.
• Immunoassays can be used to detect some enzymes directly.
◦ Measure enzyme concentration by mass
◦ e.g. creatine kinase (CK)
• Electrophoretic techniques
◦ Used to measure isoenzymes or isoforms
measurement of enzyme activity
Enzyme activity can be measured by: • Increase in product concentration • Increase in concentration of an altered coenzyme • Decrease in substrate concentration • Decrease in coenzyme concentration
A = substrate consumed ↓ produced ↑ B = coenzyme (activator) C = product D = altered coenzyme
Enzyme kinetics phases
There are 3 phases:
- Lag Phase - Sample and reagents are mixed
- Equilibrium is established
- Non-linear - Linear Phase - Product formation and substrate consumption are consistent
- Follows zero order kinetics
- This is where we measure - Substrate Depletion - Product formation has slowed
- Not linear
- Not good for measuring
- Problem if enzyme concentration is very high
enzyme kinetics
If enzyme concentration is very high in a
sample the reaction will not be linear as
you will run out of substrate.
The sample can be diluted and
reanalyzed
methods for reading enzyme reactions
• 2-point Assay (Fixed time)◦ Read at fixed times
◦ Usually an initial reading and a later reading
◦ Readings may not be during linear phase (problem)
• Kinetic Assay (Continuous Monitoring)◦ Multiple absorbance readings taken as the reaction proceeds.
(Every 30-60 sec or continuously)
◦ More accurate
◦ Deviations from linearity will be detected
zero order kinetics
- Exist during the linear phase
- The rate of reaction is only dependent on enzyme concentration
- An increase in substrate will not increase the reaction rate
- Lab enzyme procedures follow zero order kinetics
- If other variables effect rate (e.g. substrate depletion), then First-Order kinetics exist
If we are trying to measure [enzyme] then we must use excess substrate.
If there is not enough substrate, the enzyme will have nothing to act upon
Michaelis-Menton Curve
Km = Michaelis constant
= [substrate] @ ½ max velocity
Manufacturers ensure the [substrate] in enzyme
assay kits are 10-100X the Km to prevent substrate
depletion
factors that influence enzyme activity
- Substrate Concentration – make sure that substrate does not run out
- pH - enzymes have an optimal pH
- reactions should be buffered at the optimal pH - changes in pH may denature the enzyme - most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0
• Temperature - increasing the temperature will increase the enzyme activity (to a point!)
- if the temperature is increased too high the enzyme will be denatured - rate of reaction doubles for each 10 degree increase, Q10 - usual temps are 25, 30 & 37 ± 0.1oC
• Enzyme Concentration - the higher the enzyme concentration, the faster the reaction will proceed.
cofactors
- Nonproteins
- Bind to an enzyme before a reaction can occur
- Examples:
•Activators
◦ Inorganic cofactors
◦ Metals (Ca2+, Fe2+, Mg2+)
◦ Nonmetals (Br-, Cl-)
•Coenzymes
◦ Organic cofactors
◦ Phosphates, vitamins
Inhibitors & activators
• Activators - cause rate of reaction to increase
- inorganic cofactors
- required for reaction
- often metal ions (e.g. Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, K+)
or nonmetallic ions (e.g. Br- and Cl-)
• Inhibitors - cause rate of reaction to decrease
- interfere
- may be reversible or irreversible
