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
how does a competitive inhibitor work?
what is its apparent effect on Kmand on Vmax?
competes for the same binding site as the substrate
increases Km
no effect on Vmax
what is the M-M equation w/ a competitive inhibitor?
how does a noncompetitive inhibitor work?
what happens to Km?
what happens to Vmax?
doesn’t bind at substrate-binding site, so cannot be displaced by increase [S]
inhibitor allows substrate to interact normally w/ active site, but interferes w/ enzyme’s ability to catalyze rxn
Vmax is reduced (higher on Y-axis)
Km is unchanged (same on X-axis)
how does an **uncompetitive inhibitor **work?
what happens to Vmax and Km?
binds when the substrate is bound, b/c ES complex creates a binding site for the inhibitor
Vmax and Km both are reduced
with irreversible inhibition, what kind of binding does inhibitor do?
what happens to Vmax and Km?
covalent, irreversible bonding of inhibitor-enzyme
decreases Vmax
Km is unchanged
what are the effects of irreversible inhibition?
1) as duration of exposure to inhibitor increases, so does the number of inhibited enzyme molecules
2) even after inhibitor has been removed, enzyme remains inhibited (i.e. if drug is an irreversible inhibitor, must synthesize new enzyme to restore enzymatic function)
what is an effector? what kinds are there?
usually a molecule that’s part of a pathway
binds noncovalently to a subunit of a regulatory enzyme, induces a change in affinity of substrate for binding site OR alters enzymatic efficiency of active site
can be nevative or positive
what are homotropic and heterotropic modulation?
homotropic modulation: what the substrate itself is the effector; enzyme has >1 catalytic site, and binding of 1st site alters substrate affinity of remaining sites
heterotropic modulation: if effector is a molecule other than a substrate
what are allosteric enzymes?
enzymes that’re subject to regulation by effectors
often are comprised of multiple subunits
what is alpha-ALA synthase an example of?
an allosteric enzyme
its activity is under the control of an effecotr (heme)
what is aspartate carbamoyltransferase an example of?
a multimeric regulatory enzyme
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
what is unique about glucokinase re: positive cooperativity?
glucokinase has only 1 binding site glucose
enzyme assumes more open conformation upon binding a molecule of glucose, exposing active site
once glucose is phosphorylated and exits active site, the open conformation is retained briefly
this increases access to active site for subsequent glucose molecules
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
what is heme synthesis pathway an example of? explain
negative heterotropic modulation
heme inhibits g-ALA synthase, 7 steps above it in heme synthesis pathway
shows end-product inhibition/feedback inhibition
it catalyzes the **rate limiting step, **keeps level of activity in the pathway within narrow phsiological range
what is heterotropic effectors’ impact on Km and Vmax?
activators (positive effectors): decrease Km, no effect on Vmax
negative effectors: increase Km, no effect on Vmax (apparently competitive inhibition) OR Km unaffected, Vmax reduced (apparently non-competitive inhibition)
where do heterotropic effectors come from, re: signaling pathway where their effected allosteric enzyme is?
therefore what is their function?
come from outside the signaling pathway
therefore don’t maintain homeostatic control, instead serve **signaling role **
what is PKA activation by cAMP example of?
positive heterotropic modulator
cAMP binding induces dissociation of catalytic and regulatory subunits of PKA
therefore catalytic subunits can phosphorylate their substrates
what is covalent modification
addition of a modifying group to a regulatory enzyme
particularly occurs w/ Tyr, Ser, Thr, which’re substrates for phsophorylation by various protein kinases
once attach fxn group, enzymatic activity req’d to remove them
THOSE enzymes that add/remove active groups often are subj to regulation; therefore, whole process needs info from variety of metabolic, signaling pathways
what is most common form of covalent modification? what accomplishes this or undoes it?
phosphorylation/dephosphorylation
protein kinases: add phosphate group
protein phosphatases: catalyzes hydrolysis of phosphate group
is one protein regulated by one protein kinase?
no, often have many phosphorylation sites on a typical protein
a particular protein may have overlapping phosphorylation sites for different protein kinases
what is a consensus sequence?
sequence that a protein kinase/any enzyme that produces a covalent modification recognizes on its target protein
indicates that that protein has a pohsphorylation site for that kinase
what does Ca2+/Calmodulin-depednent protein kinase II (CaMKII) demonstrate?
describe binding process
positive cooperativity, Ca2+is a positive effectorof calmodulin:
Ca2+ binds calmodulin, which has 4 Ca2+binding sites
when 4 sites are bound, calmodulin undergoes conformational change from closed –> open
affinity of Ca2+for calmodulin increases as binding sites fill up
when all 4 Ca2+binding sites are filled, calmodulin is activated, can bind CaMKII
what happens when calmodulin binds CaMKII?
usually, CaMKII’s catalytic region is blocked by an inhibitory/”pseudosubstrate” region of the protein
when calmodulin binds, conformation cahgne in CaMKII moves inhibitory region away from active site
active site now accessible to substrates for CaMKII
1 substrate=itself; CaMKII phosphorylates itself, via autophosphorylation, prevents itself from returning to closed conformation even when Ca2+ levels decline and calmodulin dissociates from CaMKII = **positive homotropic effector **that acts on itself
eventually, protein phosphatase 1 removes phosphate, stops CaMKII activity
why does CamKII have a “memory”?
CaMKII is positive homotropic effector that acts on itslef by autophosphorylating & therefore keeping its active site open to phosphorylate other enzymes
it “remembers” the Ca2+signal long after binding occurred
plays key role in formation & retention of memories in the brain
eventually, protein phosphatase 1 removes phosphate from CaMKII, terminates its activity
what are zymogens?
an inactive precursor that, when proteolysed, liberates an enzyme
what are trypsinogen and chymotrypsinogen? where do they come from, what do they do?
zymogens secreted by the pancreas into the duodenum, involved in digestion
contian sequences for trypsin and chymotripsin
once in duodenum, they encounter enteropeptidase, a locally-secreted enzyme which cleaves tripsin from trypsinogen & trypsin in turn liberates chymotrypsin from its zymogen
how is the clotting pathway regulated? describe it
clotting factors are proteases that liberate enzymes that particiapte in blood coagulation
for clotting to occur, proteins that coagulate must be liberated from their zymogens
when they kick off, they rapidly induce clotting
at each step, an active protease is generated, which catalyzes the next zymogen –> active enzyme rxn
is a very highly regulated pathway so clotting cascade doesn’t activate in response to an aberrant signal
what is an enzyme cofactor?
what is its function?
many enzymes require an add’l small molecule, cofactor, to peform their catalytic fxn
sometimes the cofactor is covalently bound to the protein part of the enzyme = apoenzyme, full complex = holoenzyme
focator can be a metal ion (Fe2+, Cu2+) or organic molecule = coenzyme, (vitamin or derived from vitamin)
they transfer active groups from 1 substrate to another; do re-dox rxns; are generated for reuse btwn reactions
examples of important enzyme cofactors?
what are isoenzymes?
what does their existence demonstrate?
multiple enzymes that perform the same reaction
demonstrates that the same rxn might require different regulation, or different substrate concentrations, in 1 cell type or tissue than in another
what are the hexokinases’ function? what are they an example of?
hexokinases are 4 isozymes - hexokinase I, II, III, IV - that phosphorylate simple sugars
hexokinase IV = glucokinase, is diff from the other isoforms,
what makes glucokinase unique from the other hexokinase isoforms?
it uses excess glucose for glycogenesis & glycolysis
1) it is a monomer, the others are dimers
2) it is not inhibited by its product glucose-6-phosphate under physiologic conditions
3) it has a much higher Km for glucose than the other hexokinases
4) it is abundant in the liver, not ubiquitous throughout the tissues like the other hexokinases
what does glucokinases’s high Km mean re: its relationship to glucose?
b/c glucokinase is abundant in liver, its main role is to use excess glucose for glycogenesis and glycolysis
b/c no feedback inhibition, can do this even if G6P accumulates in liver cells
oppositely, if glucose is scarce, high Km means that glucokinase assures that available clucose is used by hexokinases in other tissues (ie brain) to maintain metabolic processes
what is creatine kinase?
where are its isozymes found, and why are its isozymes clinically significant?
CK is enzyme in energy metabolism, found mostly in muscle
CK is dimer of M and/or B subunits, has 3 isozymes:
1) CK1: tissues, including brain
2) CK2: heart
3) CK3: skeeltal muscle, heart
**normally, blood is almost all CK3 b/c of normal turnover of skeletal muscle; but increase in CK2 in blood is diagnostic of MYOCARDIAL INFARCTION in a pt who has chest pain **
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
what is the specificity constant? what does it explain?
what does a large / a small value indicate?
Kcat/Km
allows us to compare enzyme efficiency under physiological conditions
indiciates **how effecient the enzyme is when free binding sites are abdundant **
if large: reaction can proceed at high rate even if [S] is low or enzyme isn’t highly expressed
what is catalytic perfection?
Kcat/Km must be between 108 and 109 M-1s-1
if an enzyme’s Kcat/Km is close to this, enzyme is **catalytically perfect **
boundaries exist b/c diffusion rate of molecules in aqueous solution dtermines the minimum time req’d for binding sites to be vacated and reoccupied
what are the 6 categories of enzymes?
oxidoreductases
transferases
hydrolasese
lyases
isomerases
ligases
what do oxidoreductases do
transfer electrons (hydride ions or H atoms)
what do transferases do
group-transfer reactions
what do hydrolases do
hydrolysis reactions (transfer of functional groups to water)
what do lyases do
addition of groups to double bonds, or formation of double bonds by removal of groups
what do isomerases do
transfer of groups within molecules to yield isomeric forms
what do ligases do
formation of C-C, C-S, C-O, C-N bonds by condensation reactions coupled to ATP cleavage
are all enzymes proteins?
no
RNAs that catalyze reactions, ribozymes, exist
i.e. ribosomal RNAs (rRNA) catalyze many steps of protein synthesis
much more ancient than enzymes
ribozymes are not subj to high degree of regulation as in many protein enzymes
what’s the most important/complex ribozyme?
the ribosome
where mRNA translation happens, almost entirely by ribosomal RNA, w/ ribosomal proteins playing only supportive role
