Enzymes Part 1 Flashcards
The 6 classes of enzymes:
- oxidoreducatases
- transferases
- hydrolases
- lyases
- isomerases
- ligases
Oxidoreducatases catalyze:
ox-redox reactions
Transferases catalyze:
transfer of C-, N-, or P- containing groups
Hydrolases catalyze:
cleavage of bonds via the addition of water
Lyases catalyze:
cleavage of C–C, C–S, and certain C–N bonds,
Isomerases catalyze:
racemization of optical or geometric isomers
Ligases catalyze:
formation of bonds between carbon and O, S, N coupled to hydrolysis of high-energy phosphates
Enzymes are:
- protein catalysts that increase the velocity of a chemical reaction, and are not consumed during the reaction
- NOTE: some RNA can act as catalysts - these are called ribozymes
General Enzyme Properties:
- proteins
- catalysts (are regenerated)
- highly specific
- able to be regulated
- Do not change overall free energy of reaction
The set of enzymes made in a cell determines …
which metabolic pathways occur in that cell.
A holoenzyme is:
an active enzyme bound with its nonprotein component
An apoenzyme is:
an enzyme not bound to its nonprotein component that is inactive
Cofactors:
a nonprotein component required by an enzyme to function that is a metal ion such as Zn2+ or Fe2+
Coenzymes:
- a nonprotein component required by an enzyme to function that is a small organic molecule
- mostly derived from vitamins
- Can be:
- co-substrates (transiently bound; dissociate from enzyme during altered state)
- prosthetic groups (permanently bound)
Cosubstrate:
- a coenzyme that is only transiently associated with an enzyme
- dissociates from the enzyme during an altered state
- non-covalently bound to enzyme
Prosthetic Group:
- a coenzyme that is permanently associated with an enzyme and returned to its original form
- covalently bound to enzyme
Can cofactors/coenzymes limit rate of reactions?
Yes:
- if not enough is available, it will limit the amount of active anzymes
- some cofactors/coenzymes need to be regenerated at the end of a reaction. Reaction rates can slow during regeneration.
Cofactors provide functionalities that are not found in…
- the natural amino acids
- cofactors = small metal ions
NOTE: cysteine is the only amino acid that can participate in ox/redox reactions.
Isozymes:
- Have different primary structures or amino acid sequences, but catalyze the same chemical reaction and act upon the same substrate(s)
- allow for fine tuning of metabolic need based on the specific tissue
Isozymes have different:
- kcat and Km values and temperature and pH dependencies
Measurement of isozyme levels can aid in:
- diagnosis of certain diseases/injuries
- i.e. LDH H4 and MB creatinine kinase levels after myocardial infarction
Lock and Key Model:
- predefined active site
- states that enzymes and substrates must fit together like a lock and key in order for the reaction to occur/be catalyzed.
- model has been replaced by induced fit model
An enzyme Active Site is:
- a 3D cleft (pocket) formed by catalytic amino acids that come from different parts of the protein sequence
- the catalytic amino acid side chains line the active site and participate in substrate binding and catalysis
Catalytic amino acids:
- line the active site of an enzyme
- participate in substrate binding and catalysis
- are not sequential in the amino acid sequence of an enzyme - come together to form the active site after protein folding occurs to form the tertiary structure.
In comparison to the total enzyme structure, the active site occupies:
- a small volume of the total enzyme
- Amino acids not involved in the active site provide the scaffolding/stabilization of the active site
- ensures stable conformation of the active site
Mutations in the catalytic amino acids of the active site are often:
- fatal
- mutations in the non-catalytic amino acids often lead to disease, but are not immediately fatal. They cause changes to the active site conformation and, therefore, the overall enzyme function
Binding at the active site is:
- reversible
- both substrate and product can bind to the active site
Substrate and active site amino acids interact:
- non-covalently through:
- hydrogen bonds
- hydrophobic interactions
- ionic bonds
- dipole-dipole bonds
- all of these weak interactions add up to allow tight enzyme-substrate binding
The stronger the enzyme-substrate interaction, the:
- longer the substrate stays in the active site of the enzyme
- this can affect the reaction rate, as product may not easily dissociate/eject from the active site
Character of the active site:
- nonpolar
- this enhances the substrate binding by increasing electrostatic interactions
The noncovalent interactions of the active site work:
- cooperatively
- i.e. the reaction will not occur unless all possible noncovalent interactions are met
- explains why glucokinase only binds glucose and not galactose
Lock and key model is inadequate in explaining:
- enzyme specificity
- lock and key model does not explain why water does not interact with certain enzymes
Induced Fit Model:
- flexible active site
- only a specific substrate binds to the enzyme active site and induces a conformational change in the enzyme.
- This stabilizes the active conformation of the enzyme (strong binding/affinity and fast rate)
In the induced fit model, a specific substrate activates the enzyme through:
- inducing a conformational change in the enzyme that:
- orients catalytic groups on enzyme
- tighter transition state binding (more stabilized transition state)
- excluding H2O (if not a reactant)
Enzymes can increase rates of reaction:
106 to 1017 fold
Reaction rates are inversely proportional to:
the activation energy of the reaction
Enzymes create a new reaction pathway with a lower activation energy through:
- specific binding to the transition state structure
- this stabilizes the transition state structure, lowering its energy, which allows more substrate to reach its energy level.
Enzymes accelerate rates of reactions by:
- stabilizing the transition state, which lowers the energy of the transition state and makes it more feasible for substrate to reach it.
Enzyme active sites have tighter binding for _____ rather than ______.
- tighter binding for the transition state rather than the substrate.
All the catalytic amino acid binding ability is utilized at the time of:
- transition state formation (maximum stabilization).
- Only a few of the catalytic amino acids bind to the substrate.
Enzymes do not change the ____ of a reaction.
- thermodynamics/free energy/equilibrium
- enzymes only increase the rate of reaction and decrease the time in which equilibrium is reached
The three things that affect enzyme reaction velocity:
- substrate concentration
- pH
- temperature
Enzyme rate of reaction and substrate concentration:
- rate increases with increasing [S] until Vmax is reached.
- Vmax = point where all enzyme active sites are saturated with substrate.
Enzyme rate of reaction and temperature:
- initial increases in temperature increase the rate of reaction by causing more collisions between E and S
- drastic temperature increases will denature the enzyme, and reaction rate will fall
- mutations can decrease the thermal stability of the enzyme
Enzyme rate of reaction and pH:
- At extremes of pH, the enzyme is irreversibly denatured and activity is permanently lost
- pH affects the ionization of enzyme functional groups involved in catalysis or substrate binding (side chains involved in the active site) and can alter the substrate’s ionization state.
Keq =
[P]/[S]
tells us how much substrate and product will be present at the equilibrium of a reaction
The two catalytic strategies all enzymes use:
- proximity
- transition state stabilization
Catalytic Strategy: Proximity
- substrates need to 1) collide and 2) orient in the right positions. Enzymes bring together the substrates in the proper orientation, allowing the reaction to occur.
Catalytic Strategies of enzymes (5):
- Proximity (all enzymes employ this strategy)
- Transition state stabilization (all)
- Covalent catalysis or nucleophilic catalysis (some)
- General acid-base catalysis (most)
- Metal ion catalysis (many)
A potent nucleophile at the active site of an enzyme is created via:
acid-base catalysis
Catalytic triad in chymotrypsin protease:
- serine, histidine, and aspartate
- serine becomes a nucelophile, aided by histidine
- if you mutate one amino acid in the catalytic triad, you affect reaction rate and enzyme activity
Oxyanion hole:
- a space in the enzyme active site ready to bind a negatively charged group
- In chymotrypsin, the oxyanion hole stabilizes the tetrahedral intermediate. The active site has more interactions with the tetrahedral intermediate than does the substrate. Allows the reaction to move forward.
General Base:
a catalytic amino acid that can extract a proton
General Acid:
a catalytic amino acid that can accept a proton
What is the favorite amino acid for general acid/base catalysis?
- hisitidine
- pKa near physiological pH, so can act as both an acid and a base
