Lecture 8A Flashcards
What cell processes are proteins necessary for?
DNA replication, RNA transcription, protein translation
Name some structural proteins
- collagen, keratin, pilin, cytoskeletal proteins (actin, tubulin)
Name some proteins for mobility
kinesin, dynein, myosin
molecular motors
Name a protein that is a receptor
Insulin receptor
Name a protein that is a ligand
Insulin
Name some proteins in the immune system
Antibodies, complement proteins
Name some housekeeping proteins
Chaperones
Name some proteins for signalling
Kinases, phosphatases, ubiquinases
Name some proteins that are enzymes
Proteases, reductases, glucosidases
Name some proteins for storage and transport
Hemoglobin
All enzymes are proteins except which?
Except catalytic RNAs (ribozymes)
Most enzymes do what?
Catalyze the transfer of electrons, atoms, or functional groups
Enzymes classified according to what?
according to the type of transfer reaction, the group donor
and the group receptor.
Enzymes enhance rates of reaction by lowering what?
DG‡ aka the activation
energy for the transition state ‡
What are 3 strategies for enzyme catalysis?
- Enzyme binds to 2 substrate molecules and orients them precisely to encourage a reaction to occur between them
- Binding of substrate to enzyme rearranges electrons in the substrate, creating partial neg and pos charges that favour a rxn
- Enzyme strains the bound substrate molecule, forcing it toward a transition state to favour a rxn
Where do substrates bind on an enzyme?
A substrate binding pocket or groove aka the active site
Which AA’s in the enzyme’s active site participate in catalysis? What are the others used for
Residues that are distant from each other in the primary sequence but come together in the 3D folded (tertiary) structure of the protein.
Others are required for
binding to the substrate and/or the transition state,
positioning substrates such that they can react, etc
Why is the enzyme so big?
To provide a folding
framework for the active site; precisely aligns the
active site residues
The bonds between what lower DG‡?
bonds formed between enzyme and substrate
What bonds do the active site AA’s form and with what?
form multiple weak, non-covalent bonds (electrostatic, hydrogen bonds, van der Waals interactions, hydrophobic interactions) with the substrate, especially with the transition state
What are cofactors?
metal ions that are essential for the folded structure of the protein (enzyme) and/or for catalysis by orienting the substrates for reaction or stabilize the charged transition state
may also mediate oxidation-reduction
reactions by reversible changes in the metal ion’s oxidation state
What is a cofactor that is permanently attached to the enzyme?
Zn2+ in carboxypeptidase A
What is a cofactor that is not permanently attached to the enzyme and assists catalysis indirectly?
Mg2+ in ATP hydrolysis
What are coenzymes? Function? Example?
complex organic compounds that cannot be synthesized by the cell - must be taken
up in the diet – e.g., vitamins
- provide a functional group that is involved in the catalytic reaction
What are prosthetic groups?
cofactors and coenzymes that are tightly bound to enzymes or other proteins. e.g., hemoglobin.
What is the rate of a reaction (V) measured as?
as the change in reactant or substrate concentration [S] or product concentration [P] over time
What does V (rate of rxn) depend on?
concentration of the substrate and the rate constant, k, for the reaction
What is k? Units?
proportionality
constant, called the rate constant.
k is units of reciprocal time (sec-1)
Units for V?
units of M/sec (molar/sec
or moles/[litresec])
The larger the activation energy DG‡, the ___ k is?
Smaller
Does V0 increase or dec with increasing initial [S]
Increases
How to determine initial velocity, V0
amount of product formed at different substrate concentrations ([S]n) is plotted as a function of time. Enzyme concentration is kept constant and is usually far below [S] ([S]»[E]). The initial velocity (V0) for each substrate concentration is determined from the slope of the curve at the beginning of a reaction, when the reverse reaction is insignificant.
What does Michaelis Menten propose?
a simple model to account for the non-linearity of the V vs. [S] curve. This model took into account the enzyme-substrate complex (ES) as a necessary intermediate in catalysis
Is E + S -> ES or ES -> E + P the rate-limiting step?
ES -> E + P
As more substrate is added what happens to the active sites?
all of the active sites on the enzyme become
saturated with substrate - the enzyme is working at full capacity.
What is the steady state assumption?
we will use conditions where [S]»_space; [E], so [ES]
remains constant and rates of formation and breakdown of [ES] are equal
What is the Michelis-Menton equation ?
a statement of the quantitative relationship between then initial velocity, V0, the maximum velocity, Vmax, and the initial substrate concentration, [S], all related through the Michaelis constant, KM.
At half-maximal velocity, (Vmax/2), KM = ?
[S]
What is KM?
the substrate concentration that yields half-maximal velocity.
KM and Vmax values depend on ?
the enzyme and its interaction with a particular substrate
The maximal rate, Vmax, is attained when ?
the catalytic sites on the enzyme are saturated w/ substrate – this occurs at high [S].
when [S] «_space;KM the rate (V0) is ?
directly proportional to [S] (the curve is linear)
when [S]»_space; KM, [S]/([S]+[KM]) = 1 so V0 =
Vmax and the rate is maximal
when [S] = KM, Vo =
Vmax/2, then the reaction rate is at half its maximal value
Simplistically, a low KM value means that an enzyme?
binds to a substrate tightly.
A high KM value generally means that an enzyme binds
its substrate weakly
What is the turnover number of an enzyme?
“the number of substrate molecules converted to product in a unit of time for a single enzyme molecule when the enzyme is fully saturated”
Vmax is proportional to which kinetic constant?
K2
What is kcat?
rate constant or turnover number of the reaction when the enzyme is saturated with substrate (i.e., when [S]»_space; KM).
An enzyme’s effectiveness depends on?
its ability to bind substrate (affinity) and how rapidly it converts substrate to product (high turnover). kcat/KM takes into account both the binding and catalytic events.
Highly efficient enzymes bind to their substrates tightly (low KM) and process them rapidly (high kcat). These enzymes will have a high kcat/KM ratio.
Enzyme catalysis is limited by ___ rate, which is between 10^8 and 10^9 sec^-1 M^-1.
“Diffusion”
Enzymes can only process substrates at the rate at which they encounter them in solution. Some enzymes approach perfection in that they catalyze reactions at the diffusion rate - every collision is productive
What enzymes do not obey Michaelis-Menten kinetics
Allosteric enzymes
What is allosteric control?
Molecules can exert allosteric control over an enzyme by binding at one site and inducing a conformational change in the protein that alters its substrate binding/processing
Is allosteric regulation pos or neg?
Can be both
Example of neg allosteric regulation
Feedback inhibition
How many active sites do allosteric enzymes have
More than one
What is cooperatve binding? What kind of plot results from it? Pos or neg allosteric regulation?
Binding of substrate to one active site that positively influences binding to another active site
results in a
sigmoidal plot for V0 vs. [S], rather than a
hyperbolic one
pos allosteric regulation
How do small molecules and ions affect substrate binding?
Small molecules and ions can bind reversibly to an enzyme and inhibit substrate binding or substrate processing
What is competitive inhibition?
an inhibitor resembles the substrate and binds to the enzyme active site, thereby blocking substrate binding. The competitive inhibitor can be displaced by increasing the substrate concentration (i.e. the substrate competes with the inhibitor).
What is Non-competitive inhibition?
an inhibitor binds to a site on the enzyme that is distinct from the active site, and exerts a negative allosteric effect on substrate binding. The substrate can still bind but the enzyme cannot convert it to its transition state as efficiently, slowing or preventing catalysis. Non-competitive inhibition cannot be overcome by increasing the substrate concentration.
What are irreversible inhibitors?
bind very tightly to an enzyme, either covalently or non-covalently and inactivate them. In most cases these inhibitors interact with the enzyme active site, rendering the enzyme inactive. (e.g., DFP, which covalently binds to active site serines.) Almost all irreversible inhibitors are toxic.
Irreversible inhibitors typically will bind to more than one enzyme within an enzyme class.
What are substrate analogs?
resemble the substrate, can bind to the enzyme, but can not be “turned over” (turned to product)
What are transition state analogs
Transition state analogs are inhibitors that resemble the transition state of a substrate. They also cannot be turned over. Substrate and transition state analogs are often used to crystallize enzymes to solve their atomic structures and understand their reaction mechanisms.
An example of irreversible inhibitor?
Diisopropyl fluorophosphate (DFP) reacts with a catalytically important serine group in an enzyme active site to form a covalent adduct. The serine is no longer active; the adduct may also block substrate binding.
