LEC9-11: Enzymes & Enzyme Kinetics Flashcards
what does it mean that biochemical reactions in the body are thermodynamically favored?
free energy (G) of the products is lower than that of the reactants
aka ΔG has a **negative **value
if combined free energy of X+Y is less than that of A+B, which way is this reaction favored?
A+B –> X+Y
favored in direction of X+Y
what is the formula for Keq of the reaction A + B –> X + Y?
when is this reaction thermodynamically favored and spontaneously occurring?
Keq = [X][Y] / [A][B]
thermodynamically favored & spontaneously occurring when Keq < 1
what are transition states? what do they do to rates of rxns? how are they surpassed?
transition states: high-energy intermediates that are barriers to spontaneous reaction occurring
cause highly favorable reactions to proceed at very low rates spontaneously
energy required to each one is G+, its activation energy
how do enzymes interact with reactants in a chem rxn? what is the consequence?
enzymes breifly bond w/ reactants (substrates) **in their transition states **at the **active site **of an enzyme
these reactant-enzyme complexes have low free energy compared to transition state of non-catalyzed reaction
thus enzymes accelerate the reaction
how does the enzyme chymotrypsin work?
lowers the transition states’ energy in the rxn btwn H2O + a polypeptide
1st peak: energy to strip peptide of water molecules in the aq environment, allows initial bond form btwn substrate and enzyme
this bond forms, results in drop in energy
2nd peak: first of 2 transition states catalyzed by chymotropin
3rd, tallest peak: maximum energy, assoc w/ 2nd transition state
with an enzyme present, what changes - the activation energy, or the net energy? how is change?
with the enzyme present,
activation energy for catalyzed reaction is smaller than for the spontaneous reaction so reaction occurs,
even though
**net energy change **is the same for catalyzed and spontaneous reaction
what is this curve showing
effect of enzyme-mediated catalysis on energy profile of a reaction:
enzyme lowers the transition states for the reaction
net energy change remains the same for catalyzed and spontaneous reaction
do enzymes impact equilibrium of reaction?
why/why not?
enzymes DO NOT affect equilibrium of the reaction
equilibrium depends solely on ΔG
ENZYME CHANGES ONLY THE REACTION RATE
what is the enzyme dogma?
to what extent does it occur?
ENZYME ONLY CHANGES THE REACTION RATE
THEY DO NO AFFECT THE EQUILIBRIUM OF THE REACTION
however, this change can be so dramatic that it can be like an “off” to “on” switch for a rxn:
usu enzymes increase rate by factor of 106-1014
a rxn that’d spontaneously occur 1x/yr could, w/ enzyme catalysis, occur 30x/millisecond (1011-fold increase)
what would happen in this reaction if B is continually depleted?
rate of A –> B increases
per Le Chatelier’s principle (change in concentration of a reactant or product will shift equilibrium of reactions involved)
what is a “uni-uni” reaction
rxn in which a single reactant (substrate) transformed into single product
i.e. when an isomerase catalyzes the conversion btwn stereoisomers
what is a “bi-bi” reaction?
i.e. phosphotransferase, transfers a phosphate group from 1 substrate to another
2 substrates, 2 products
what is a “bi-uni” reaction?
2 substrates joining together into 1 product
i.e. by a ligase
where is an enzyme’s active site?
what happens there?
enzyme binds substrate in its active site
active site lies in a groove or pocket of the enzyme, typically includes residues from different segments of the polypeptide chain
what kind of reaction occurs between chymotrypsin and phenylalanine?
bi-bi
what is the binding site of the lock-and-key model?
**when the binding site is optimized for the substrate **
there’s a relatively stable region of an enzyme for its corresponding reactant
recognizes the reactant based on characteristics like its topology, electrostatic profile, potential to form bonds w/ amino acids in the active site
what is the active site in the induced-fit model?
unoccupied binding site has low affinity for the substrate, but binding induces a conformational change in enzyme that makes active site have a high affinity for substrate
this brings reactive groups of enzyme close to substrate
may be only one transition state but may need series of transition states to be induced before reaction’s complete
more accurate model of enzyme-substrate binding
in a thermodynamically favorable reaction, what is the relationship btwn energy of the reactants and energy of the products?
what must be achieved to get from reactants to products?
energy of the reactants is HIGHER than energy of the products
need activation energy to attain the transition state, when bond has been made but not cleaved; transition state energy > reactant energy
how does the substrate react to its enzyme in the induced-fit model?
**requires substrate to undergo steric changes that bring it closer to a transition state **
often, there are several transition state induced before rxn is complete
these conformational changes of enzyme bring other regions of the protein close to the substrate; they interact, or places multiple subtrates near each other in an orientation to favor a rxn
describe the relationship between glucose and glucokinase
what kind of enzyme-substrate binding is this?
induced-fit model
glucokinase (a hexokinase) catalyzes the phosphorylation of glucose
glucokinase has 2 domains, joined by a hinge region
when unbound, glucokinase is open conformation
when glucose binds, glucokinase is **closed, & thus catalytically active **
why does the induced fit model make more thermodynamic sense than the lock and key model?
lock-and-key provides a large decrease in G upon binding (ES), but reaching transition state from this low energy would be rare
induced fit, have small decrease in G upon binding (ES), but reaching transition state is greatly reduced compared to spontaneous, b/c enzyme-transition state complex has **much lower G than transition state alone. **reaction proceeds more rapidly.
what is enzyme kinetics
the study of the rate of enzymatically catalyzed rxns as a fxn of variables that include substrate & enzyme concentration, drugs that inhibit enzyme fxn, temperature, pH, etc.
how is the rate of rxn of substrate –> product measured?
measured when the rxn is initiated, by adding substrate to enzyme
measure conversion of substrate –> product before product accumulates & reverse rxn becomes significant
what is the inital rate of a substrate –> product rxn called?
initial velocity, V0
what does this show? explain!
for most enzymes, V0, initial velocity, is a hyperbolic function of [S]
when [S] is low, V0 is proportional to [S] = first-order kinetics
when [S] is high, V0 reaches asymptote, Vmax and becomes independent of [S] = zero-order kinetics
what is first-order vs. zero-order reaction w/ enzyme-substrate relationship?
first-order: when V0, initial rate, is directly proportional to [S]
zero-order: when [S] is very high, rate is independent of [S]; relationship flattens, and V0 becomes constant
what is Vmax?
highest possible reaction rate for a given catalyzed reaction
what is Km?
what does it mean if the reaction’s Km is lower w/ 1 enzyme than another?
Km = substrate concentration at which V0 is 50% of Vmax
aka affinity of the substrate for the enzyme
lower Km = higher affinity; higher Km = lower affinity
which enzyme has a lower Km, a higher affinity, and what is the relationship of their Vmax?
purple: lower Km so higher affinity substrate
same Vmax though of both substrates w/ the enzyme, even though affinities differ
what is the michaelis-menten equation?
describes hyperbolic relationship between [S] and Km of enzyme-substrate
Km is the Michaelis constant
if [S] < Km, V0 is directly proportional to [S] = first-order behavior
if [S] > Km, V0 = Vmax = zero-order behavior
state the lineweaver-burke transformatin of the michaelis-mentin equation
name the variables
slope = Km / Vmax
y-intercept = 1/Vmax
what is the M-M equation w/ a competitive inhibitor?
what is positive cooperativity? what does it look like on a graph?
what is it an example of?
homotropic modulation
1st site’s occupation increases affinity of subsequent sites to bind
V0 vs [S] is sigmoidal (i.e. hemoglobin)
describe model of homotropic allostery
enzyme is a homomer of 2 subunits, each can either be in relaxed or taut state
R=high affinity; T=low
both subunits initially are in T
when substrate binds 1 subunit, it induces a fit/creates a high-affinity site, converts that subunit to the R conformation
now other subunit also assumes R conformation, presenting a high-affinity binding site
how does positive heterotropic modulation work?
binding to a site on a regulatory subunit (R) increases affinity of catalytic subunit (S) for substrate
effectors here don’t covalently modify the enzyme - they bind, induce conformational change, and dissociate
usually binding molecule is the rate limiting step for the pathway
examples of important enzyme cofactors?
what is kcat? what does it measure?
the turnover number of an enzyme: the number of operations that a single molecule of an enzyme can perform per second when the enzyme is saturated
allows us to compare the efficiencies of different enzymes
