Ch. 2: Enzymes Flashcards
what is the most important function of enzymes?
incredibly important as biological catalysts
do catalysts impact the thermodynamics of a biological reaction?
no, so deltaHrxn and equilibrium position do not change
func: catalyst
help the reaction proceed at a much faster rate
is the enzyme changed during the course of the reaction?
no, since it is a catalyst
what are the 7 key features of enzymes to remember?
- lower the activation energy
- increase the rate of the reaction
- do not alter the equilbrium constant
- are not changed or consumed in the reaction (they will appear in both the reactants and products)
- are pH and temperature sensitive, with optimal activity at specific pH ranges and temperatures
- do not affect the overall deltaG of the reaction
- are specific for a particular reaction or class of reactions
defn: substrates
the molecules upon which an enzyme acts
defn: enzyme specificity
a given enzyme will only catalyze a single reaction or class of reactions with these substrates (enzymes are picky!!)
mnemonic: what are the 6 categories of enzymes?
also what are these categories based on?
based on function or mechanism
LIL HOT
1. ligase
2. isomerase
3. lyase
4. hydrolase
5. oxidoreductase
6. transferase
if you don’t recognize an enzyme, what suffix should you keep in mind that most enzymes have?
-ase
func: oxidoreductase
catalyze oxidation-reduction reactions (the transfer of electrons between biological molecules)
what is often paired with oxidoreductases? what is the function of this? what are 2 examples of this?
often paired with a cofactor
which acts as an electron carrier
such as NAD+ or NADP+
defn: reductant, oxidant
REDUCTANT = the electron donor in reactions catalyzed by oxidoreductase
OXIDANT= the electron acceptor in reactions catalyzed by oxidoreductase
what are the 2 words in enzyme names that usually signal oxidoreductase?
- dehydrogenase
- reductase
what does oxidase usually signal in enzyme names?
oxygen is the final electron acceptor
func + naming: transferase
catalyze the movement of a functional group from one molecule to another
usually very straightforwardly named
what group of enzymes do kinases belong to?
transferases!
func: kinase
catalyze the transfer of a phosphate group, generally from ATP, to another molecule
func: hydrolase
catalyze the breaking of a compound into 2 molecules using the addition of water
naming + 4 examples: hydrolases
typically named only for their substrate
- phosphatase –> cleaves a phosphate group from another molecule
- peptidase –> breaks down proteins
- nuclease –> breaks down nucleic acids
- lipase –> breaks down lipids
func: lyase
catalyze the cleavage of a single molecule into two products
do lyases require water as a substrate?
do lyases act as oxidoreductases?
no and no!
can most enzymes catalyze the reverse of their specific reactions? what does this imply about lyases?
yes!
the synthesis of two molecules into a single molecule may also be catalyzed by a lyase
defn: synthases
what a lyase is referred to when it is fulfilling the function of synthesizing two molecules into a single molecule
func: isomerase
catalyze the rearrangement of bonds within a molecule
can isomerases be classified as other types of enzymes? if yes, what types of enzymes and why?
yes
oxidoreductases, transferases, or lyases –> depends on the mechanism of the enzyme
do isomerases catalyze reactions between stereoisomers or constitutional isomers?
both!
func: ligase
catalyze addition or synthesis reactions, generally between large similar molecules, and often require ATP
what is the difference in functionality between a ligase and a lyase (acting as a synthase)?
synthesis reactions with large molecules –> ligase
synthesis reactions with smaller molecules –> lyase
when are ligases most likely encountered on test day?
in nucleic acid synthesis and repair
what is the main role of thermodynamics?
relates the relative energy states of a reaction in terms of its products and reactants
defn: endergonic vs. exergonic reactions
endergonic reaction = one that requires energy input (delta G > 0)
exergonic reaction = one in which energy is given off (delta G < 0)
do enzymes affect equilibrium?
no, but they can affect how quickly a reaction GETS to equilibrium
what is the functional consequence of the fact that enzymes, as catalysts, are unchanged by the reaction?
far fewer copies of the enzyme are required relative to the overall amount of substrate because one enzyme can act on many, many molecules of substrate over time
how do catalysts exert their effect?
by lowering the activation energy of a reaction
they make it easier for the substrate to reach the transition state
why is the reversal of most reactions catalyzed by enzymes essentially nonexistent?
although they are TECHNICALLY reversible, that reversal may be extremely energetically unfavorable and thus nonexistent
analogy: how catalysts exert their effect
imagine having to walk to the other side of a tall hill
the only way to get there is to climb to the top of the hell and then walk down the other side, but wouldn’t it be easier if the top of the hill was cut off so one wouldn’t have to climb so high?
that is what catalysts do for substrates to achieve their transition state
enzyme mechanisms will vary depending on the reaction being catalyzed, but they tend to share some common features. what are these 3 common features?
- act to provide a favorable microenvironment in terms of charge or pH
- stabilize the transition state
- bring reactive groups nearer to one another in the active site
what is the key catalytic activity of an enzyme? what impact does this have?
key activity: the formation of the enzyme-substrate complex in the active site of an enzyme
impact: reduces the activation energy of the reaction
what does the interaction between a substrate and the active site of an enzyme account for in general terms?
the selectivity and some regulatory mechanisms of enzymes
defn: substrate
the molecule upon which an enzyme acts
defn: enzyme-substrate complex
the physical interaction between the enzyme and the substrate
defn: active site
the location within an enzyme where the substrate is held during the chemical reaction
what dictates the specificity of a specific enzyme for a molecule or group of molecules?
the active site assumes a defined spatial arrangement in the enzyme-substrate complex which dictates this
what 3 things within the active site stabilize its spatial arrangement, and thus contribute to the efficiency of the enzyme?
- hydrogen bonding
- ionic interactions
- transient covalent bonds
what are the 2 competing theories explain how enzymes and substrates interact? which is more scientifically accepted?
- lock and key theory
- induced fit model (more scientifically accepted)
explain (2): lock and key theory
- the enzyme’s active site (lock) is already in the appropriate conformation for the substrate (key) to bind
- the substrate can then easily fit into the active site, like a key into a lock or a hand into a glove
is alteration of the tertiary or quaternary structure necessary upon binding of the substrate in the lock and key theory?
no
explain via analogy (5): induced fit model
- imagine that the enzyme is a foam stress ball and the substrate is a frustrated MCAT student’s hand
- as the student’s hand squeezes the ball, both change conformation (the ball is no longer spherical, the hand is no longer flat) because they adjust to fit each other well
- in this case, the substrate (student) has induced a change in the shape of the enzyme (the stress ball) –> this requires energy, and is thus endergonic
- letting go of the stress ball is easy and doesn’t require extra energy, so this is exergonic
- just like enzymes, foam stress balls return to their original shape once their crushers (substrates) let go of them
explain molecularly (3): induced fit model
- starts with a substrate and an enzyme active site that don’t seem to fit together
- once the substrate is present and ready to interact with the active site, the molecules find that the induced form (transition state) is more comfortable for both of them
- thus, the shape of the active site becomes truly complementary only after the substrate begins binding to the enzyme
what happens in the induced fit model if a substrate of the wrong type is present? (2)
- a substrate of the wrong type will not cause the appropriate conformational shift in the enzyme
- thus, the active site will not be adequately exposed, the transition state is not preferred, and no reaction occurs
diagram comparison: lock and key theory vs. induced fit model
func: cofactors or coenzymes
required by many enzymes to be effective
char (3): cofactors or coenzymes
- nonprotein molecules
- small in size
- attached to their enzymes in a variety of ways, ranging from weak noncovalent interactions to strong, covalent ones
why are cofactors/coenzymes typically small in size?
so they can bind to the active site of the enzyme and participate in the catalysis of the reaction, usually by carrying charge through ionization, protonation, or deprotonation
why are cofactors/coenzymes usually kept at low concentrations in cells?
so they can be recruited only when needed
defn: apoenzymes vs. holoenzymes
APOenzymes = enzymes without their cofactors
HOLOenzymes = enzymes containing their cofactors
defn: prosthetic groups
tightly bound cofactors or coenzymes that are necessary for enzyme function
what are cofactors typically?
inorganic molecules or metal ions, often ingested as dietary materials
what are coenzymes typically?
small organic groups, the vast majority of which are vitamins or derivatives of vitamins such as NAD+, FAD, and coenzyme A
what vitamins are included in water-soluble vitamins? (2)
- B complex vitamins
- ascorbic acid (vitamin C)
why must water-soluble vitamins be replenished regularly?
they are important coenzymes that are easily excreted
what are the fat-soluble vitamins? (4)
- A
- D
- E
- K
what are the fat-soluble vitamins regulated by?
partition coefficients, which quantify the ability of a molecule to dissolve in a polar vs. nonpolar environment
are enzymatic reactions restricted to a single cofactor or coenzyme?
no, many metabolic reactions require multiple!
what two basic factors affect enzyme kinetics?
- environmental conditions
- concentrations of substrate and enzyme
explain why the concentrations of the substrate [S] and enzyme [E] greatly affect how quickly a reaction will occur (5)
- let’s say we have 100 stress balls (enzymes) and only 10 frustrated students (substrates) –> high [E] relative to [S]
- because there are many active sites available, we will quickly form products (students feeling relaxed) and reach equilibrium quickly
- as we slowly add more substrate (students), the rate of the reaction will increase (more people will relax in the same amount of time because we have plenty of available stress balls)
- however, as we add more and more people (and start approaching 100 students), we begin to level off and reach a maximal state of “relaxation”. there are fewer and fewer available stress balls until finally all active sites are occupied
- now, inviting more students into the room will not change the rate of the reaction (it cannot go faster once it has reached saturation)
defn: vmax
the maximum velocity that the enzyme works at
what is the only way to increase vmax?
how can this be accomplished?
increasing the enzyme concentration
can be accomplished by inducing the expression of the gene encoding the enzyme
diagram + brief explanation: Michaelis-Menten Plot of Enzyme Kinetics
as the amount of substrate increases, the enzyme is able to increase its rate of reaction until it reaches a maximum enzymatic reaction rate (vmax)
once vmax is reached, adding more substrate will not increase the rate of reaction
what does the Michaelis-Menten equation describe for most enzymes?
how the rate of the reaction, v, depends on the concentration of both the enzyme [E], and the substrate, [S], which forms product [P]
defn: k1
the rate at which enzyme-substrate complexes form
defn: k-1
the rate of dissociation of the ES complex (into E + S)
defn: kcat
the rate that the ES complex turns into E + P
relationship utilizing k1, k-1, and kcat
is the [E] constant?
on test day, yes!
eqn: Michaelis-Menten equation
what does the Michaelis-Menten equation turn into when the reaction rate is equal to half of vmax?
defn + name + func: Km
the substrate concentration at which half of the enzyme’s active sites are full
the Michaelis constant
often used to compare enzymes
what else is Km a measure of under certain conditions?
the affinity of the enzyme for its substrate
what does a higher Km mean when comparing two enzymes, if we consider Km as a measure for the affinity of the enzyme for its substrate? why does it mean that?
higher Km = lower affinity for its substrate
why? it requires a higher substrate concentration to be half-saturated
can Km be altered? why or why not?
No, it is an intrinsic property of the ES system and cannot be altered by changing the concentration of substrate or enzyme
for a given concentration of enzyme, what is the shape of a graph of the Michaelis-Menten relationship typically?
a hyperbola
what affect will changes in substrate concentration have on the reaction
when substrate concentration is less than Km? exceeds Km?
less than Km: changes in substrate concentration will greatly affect the reaction rate
higher than Km: the reaction rate increases much more slowly as it approaches vmax, where it becomes independent of substrate concentration
what does vmax represent and what is its unit?
vmax = maximum enzyme velocity
unit: moles of enzyme per second
eqn + units: mathematically relating vmax to kcat
unit: s^-1
what does kcat measure qualitatively?
the number of substrate molecules “turned over” or converted to product, per enzyme molecule per second
most enzymes have kcat values within what range?
101 - 103
how can we rearrange the Michaelis-Menten equation to include kcat?
what can the Michaelis-Menten derived equation be simplified to at very low substrate concentrations (where Km»_space; [S])?
defn: catalytic efficiency of the enzyme
the ratio of kcat/Km
how are 2 ways we obtain a high catalytic efficiency?
- a large kcat (high turnover)
- a small Km (high substrate affinity)
what is a Lineweaver-Burk plot?
a double reciprocal graph of the Michaelis-Menten equation
what do the intercepts of the line with the x and y axis represent on a Lineweaver-Burk plot? + diagram
x-axis intercept = -1/Km
y-axis intercept = 1/vmax
for what and why is a Lineweaver-Burk plot particularly useful?
determining the type of inhibition that an enzyme is experiencing because vmax and Km can be compared without estimation
why do certain enzymes show sigmoidal (S-shaped) kinetics? + diagram
owing to cooperativity among substrate binding sites
char (3): cooperative enzymes
- have multiple subunits
- have multiple active sites
- subject to activation and inhibition, both competitively and through allosteric sites
what are the two states that subunits and enzymes may exist?
- a low-affinity tense state (T)
- a high-affinity relaxed state (R)
what encourages transition of subunits from the T to R state?
what is the impact of this transition?
binding of the substrate encourages
the transition increases the likelihood of substrate binding by these other subunits
what encourages transition of subunits from the R to T state?
what is the impact of this transition?
loss of the substrate can encourage
the transition promotes the dissociation of substrate from the remaining subunits
explain the analogy of thinking of cooperative enzyme kinetics like a party (2)
- as more people start arriving, the atmosphere becomes more relaxed and the party seems more appealing
- but as people start going home, the party dies down and more people are encouraged to leave so the tense hosts can clean up
what types of enzymes often show cooperative kinetics?
regulatory enzymes in pathways
defn + func: Hill’s coefficient
a way of quantifying cooperativity
indicates the nature of binding by the molecule
what does a Hill’s coefficient > 1 mean?
<1 mean?
= 1 mean?
> 1 –> positively cooperative binding is occurring, such that after one ligand is bound the affinity of the enzyme for further ligands increases
< 1 –> negatively cooperative binding is occurring, such that after one ligand is bound the affinity of the enzyme for further ligands decreases
= 1 –> the enzyme does not exhibit cooperative binding
what 3 characteristics of an environment have significant effects on the ability of an enzyme to work?
- temperature
- acidicity or alkalinity (pH)
- high salinity
what 2 terms are synonymous with enzyme activity on the MCAT?
enzyme activity = enzyme velocity = enzyme rate
how does enzyme velocity respond to increases in temperature?
enzyme-catalyzed reactions tend to DOUBLE in velocity for every 10 deg C increase in temperature until the optimum temperature is reached
what happens to the enzyme after reaching its optimum temperature?
activity falls off sharply, as the enzyme will denature at higher temperatures
can enzymes regain their function after being overheated?
some can, if they are cooled
why are Siamese cats an example of enzymes being influenced by temperature?
the enzyme responsible for pigmentation, tyrosinase, is mutated and is ineffective at body temperature, but active at cooler temperatures
so only the tail, feet, ears and face have an active form of the enzyme and are dark
what are the 2 main reasons why enzymes depend on pH to function properly?
- pH affects the ionization of the active site
- changes in pH can lead to enzyme denaturation
For enzymes that function in human blood, the optimal pH is 7.4, what are the two exceptions to this optimal pH?
- pepsin (in the stomach, has maximal activity around pH 2)
- pancreatic enzymes (in the small intestine, work best around pH 8.5)
diagram: effects of temperature and pH on rate of enzyme action
what effects (3) can salinity or osmolarity have on enzyme activity?
does this have a physiologic significance?
- increasing levels of salt can disrupt hydrogen and ionic bonds
- this causes a partial change in the conformation of the enzyme
- in some cases causes denaturation
no it does not generally have physiologic significance, but rather affects enzyme activity in vitro
defn: feedback regulation
regulation by products further down a given metabolic pathway
defn: feed-forward regulation
regulation by intermediates that precede the enzyme in the pathway
is feedback activation or feedback inhibition more common with enzymes?
feedback inhibition (negative feedback)
defn: negative feedback/feedback inhibition
once we have enough of a given product, we want to turn off the pathway that creates the product, rather than creating more
how does negative feedback work with enzymes? (2)
- the product may bind to the active site of an enzyme or multiple enzymes that acted earlier in its biosynthetic pathway
- this competitively inhibits these enzymes, making them unavailable for use
diagram: feedback inhibition by the product of a metabolic pathway
a high concentration of the product, D, inhibits enzyme 1, slowing the entire pathway
what are the 4 types of reversible inhibition?
what 2 types come up most often on the MCAT?
- competitive (common)
- noncompetitive (common)
- mixed
- uncompetitive
defn: competitive inhibition
occupancy of the active site (substrates cannot access enzymatic binding sites if there is an inhibitor in the way)
how can competitive inhibition be overcome? why does this work?
by adding more substrate so that the substrate-to-inhibitor ratio is higher
if more molecules of substrate are available than molecules of inhibitor, then the enzyme will be more likely to bind substrate than inhibitor (assuming the enzyme has equal affinity for both molecules)
how does a competitive inhibitor affect vmax? Km? why?
does NOT change vmax (if enough substrate is added, it will outcompete the inhibitor and be able to run the reaction at vmax)
INCREASES Km (the substrate concentration has to be higher to reach half the vmax in the presence of the inhibitor)
diagram: lineweaver-burk plot of competitive inhibition
defn: noncompetitive inhibition
bind to an allosteric site instead of the active site, which induces a change in enzyme conformation
because the two molecules do not compete for the same side, it is noncompetitive
defn: allosteric site
non-catalytic regions of the enzyme that bind regulators
can noncompetitive inhibition be overcome by adding more substrate? why or why not?
no
noncompetitive inhibitors bind equally well to the enzyme and the enzyme-substrate complex
once the enzyme’s conformation is altered, no amount of extra substrate will be conducive to forming an ES complex
how does a noncompetitive inhibitor affect vmax? Km? why?
DECREASES vmax –> there is less enzyme available to react
does NOT alter Km –> any copies of the enzyme that are still active maintain the same affinity for their substrate
diagram: lineweaver-burk plot of noncompetitive inhibition
defn: mixed inhibition
results when an inhibitor can bind to either the enzyme or the ES complex, but has a different affinity for each
they bind at an allosteric site, not the active site
if the inhibitor in mixed inhibition had the same affinity for both the enzyme and the ES complex, what type of inhibitor would it be equal to?
a noncompetitive inhibitor
how does a mixed inhibitor affect vmax? Km? why?
alters Km depending on the preference of the inhibitor for the enzyme vs. the ES complex
if it prefers to bind to the enzyme: INCREASES Km value (lowers affinity)
if it prefers to bind to the ES complex: LOWERS Km value (increase affinity)
either way, vmax DECREASES
setup: lineweaver-burk plot of mixed inhibition
the curves for the activity with and without the inhibitor intersect at a point that is not on either axis
defn: uncompetitive inhibition
bind only to the ES complex and essentially lock the substrate in the enzyme, preventing its release (can be interpreted as increasing affinity between the enzyme and the substrate)
why must uncompetitive inhibitors bind at an allosteric site?
because the ES complex has already formed upon binding
in fact, it is the formation of the ES complex that creates a conformational change that allows the uncompetitive inhibitor to bind
how does an uncompetitive inhibitor affect vmax? Km? why?
LOWER Km and vmax
setup: lineweaver-burk plot of uncompetitive inhibition
the curves for activity with and without the inhibitor are parallel
defn: irreversible inhibition
the active site is made unavailable for a prolonged period of time or the enzyme is permanently altered, and thus is not easily overcome or reversed
in what circumstances does irreversible inhibition often come up?
it is a prime drug mechanism!
defn: allosteric site
a site other than the active site that can regulate the availability of the active site
char: allosteric enzymes (2)
- alternative between an active and an inactive form (the inactive form cannot carry out the enzymatic reaction)
- have multiple binding sites (the active site, and at least one allosteric site)
defn: allosteric activators or allosteric inhibitors
molecules that bind to the allosteric site
func (both and individual): allosteric activators or allosteric inhibitors
binding of either causes a conformational shift in the protein, or may alter the activity of the enzyme
ACTIVATOR: results in a shift that makes the active site more available for binding to the substrate
INHIBITOR: results in a shift that makes the active site less available for binding to the substrate
what covalent modifications (3) are enzymes subject to and what affect does each have on the enzyme?
- phosphorylation (may activate or deactivate enzyme)
- dephosphorylation (may activate or deactivate enzyme)
- glycosylation (the covalent attachment of sugar moieties) –> can tag an enzyme for transport within the cell, or can modify protein activity and selectivity
defn + char (4): zymogen
inactive forms of enzymes that are particularly dangerous if not tightly controlled
- contain a catalytic (active) domain and regulatory domain
- the regulatory domain must be removed or altered to expose the active site
- most have suffix -ogen
- exhibit similar regulation to apoptoic enzymes
