Chapter 2: Enzymes

What do cofactors and coenzymes do? How do they differ?

Cofactors and coenzymes both act as activators of enzymes. Cofactors tend to be inorganic (minerals), while coenzymes tend to be small organic compounds (vitamins). In both cases, these regulators induce a conformational change in the enzyme that promotes its activity. Tightly bound cofactors or coenzymes that are necessary for enzyme function are termed prosthetic groups.

What is enzyme cooperativity?

Cooperativity refers to the interactions between subunits in a multisubunit enzyme or protein. The binding of substrate to one subunit induces a change in the other subunits from the T (tense) state to the R (relaxed) state, which encourages binding of substrate to the other subunits. In the reverse direction, the unbinding of substrate from one subunit induces a change from R to T in the remaining subunits, promoting unbinding of substrate from the remaining subunits.

Which of the following is NOT a method by which enzymes decrease the activation energy for biological reactions? A) Modifying the local charge environment B) Forming transient covalent bonds C) Acting as electron donors or acceptors D) Breaking bonds in the enzyme irreversibly to provide energy

D) Breaking bonds in the enzyme irreversibly to provide energy

In the equation below, substrate C is an allosteric inhibitor to enzyme 1. Which of the following is another mechanism necessarily caused by substrate C? A) Competitive inhibition B) Irreversible inhibition C) Feedback enhancement D) Negative feedback

D) Negative feedback

A certain cooperative enzyme has four subunits, two of which are bound to substrate. Which of the following statements can be made? A) The affinity of the enzyme for the substrate has just increased. B) The affinity of the enzyme for the substrate has just decreased. C) The affinity of the enzyme for the substrate is at the average for this enzyme class. D) The affinity of the enzyme for the substrate is greater than with one substrate bound.

D) The affinity of the enzyme for the substrate is greater than with one substrate bound.

Consider a biochemical reaction A--> B, which is catalyzed by A-B dehydrogenase. Which of the following statements is true? A) The reaction will proceed until the enzyme concentration decreases. B) The reaction will be most favorable at 0 C C) A component of the enzyme is transferred from A to B D) The free energy (ΔG) of the catalyzed reaction is the same as for the uncatalyzed reaction.

D) The free energy (ΔG) of the catalyzed reaction is the same as for the uncatalyzed reaction. While the activation energy is lowered, ΔG remains unchanged in the presence of an enzyme.

What is enzyme specificity?

Enzyme specificity refers to the idea that a given enzyme will only catalyze a given reaction or type of reaction.

effect of temperature on enzyme activity

Enzyme-catalyzed reactions tend to double in velocity for every 10 degree C increase in temperature until the optimum temperature is reached. After this, activity falls sharply as the enzyme will denature at higher temperatures.

In what ways do enzymes affect the thermodynamics vs. the kinetics of the reaction?

Enzymes have no overall effect on the overall thermodynamics of the reaction; they have no effect on ΔG or ΔH of the reaction, although they do lower the energy of the transition state, thus lowering the activation energy. However, enzymes have a profound effect on the kinetics of a reaction. By lowering activation energy, equilibrium can be achieved faster (although equilibrium position does not change)

mechanisms of enzyme activity

Enzymes may 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 feedback inhibition?

Feedback inhibition refers to the product of an enzymatic pathway turning off enzymes further back in that same pathway. This helps maintain homeostasis as product levels rise, the pathway creating this product is appropriately downregulated.

What is the ideal temperature for most enzymes in the body? The ideal pH? -Ideal temperature -Ideal pH (most enzymes) -Ideal pH (gastric enzymes) -Ideal pH (pancreatic enzymes)

Ideal temperature = 37 C = 98.6 F = 310 K Ideal pH = 7.4 Ideal pH (gastric enzymes) = 2 Ideal pH (pancreatic enzymes) = 8.5

What are the effects of increasing [S] on enzyme kinetics? What about increasing [E]?

Increasing [S] has different effects depending on how much substrate is present to begin with. When the substrate concentration is low, an increase in [S] causes a proportional increase in enzyme activity. At high [S], when the enzyme is saturated, increasing [S] has no effect on the activity because Vmax has already been attained. Increasing [E] will always increase Vmax, regardless of the starting concentration of the enzyme.

What is irreversible inhibition?

Irreversible inhibition refers to the prolonged or permanent inactivation of an enzyme, such that it cannot be easily renatured to gain function.

catalytic efficiency

Kcat/Km A large Kcat (high turnover) or a small Km (high substrate affinity) will result in higher catalytic efficiency, a more efficient enzyme.

What does Km represent? What would an increase in Km signify?

Km is a measure of an enzyme's affinity for its substrate and is defined as the substrate concentration at which an enzyme is functioning at half of its maximal velocity. (At 1/2 Vmax, [S] = Km) As Km increases, an enzyme's affinity for its substrate decreases.

types of enzymes

LIL HOT L - ligase I - isomerase L - lyase H - hydrolase O - oxidoreductase T - transferase

What are the names and main functions of the six different classes of enzymes?

Ligase - addition or synthesis reactions, generally between larger molecules; often require ATP Isomerase - rearrangement of bonds within a compound Lyase - cleavage of a single molecule into two products, or synthesis of small organic molecules Hydrolase - breaking of a compound into two molecules using the addition of water Oxidoreductase - oxidation-reduction reactions (transferring electrons) Transferase - movement of a functional group from one molecule to another

How do the lock and key theory and induced fit model differ?

Lock and Key theory: -active site of enzyme fits exactly around substrate -no alterations to tertiary or quaternary structure -less accurate model Induced Fit Model: -active site of enzyme molds itself around substrate only when substrate is present -tertiary and quaternary structure is modified for enzyme to function -more accurate model

effect of pH on enzyme activity

Most enzymes also depend on pH to function properly. pH not only affects the ionization of the active site but also because changes in pH can lead to denaturation of the enzyme.

cooperativity

SIGMOIDAL CURVE Cooperative enzymes have multiple subunits and multiple active sites. low-affinity tense state (T) --> high-affinity relaxed state (R) -usually regulatory enzymes in pathways; also subject to activation and inhibition, both competitively and through allosteric sites

What do the x- and y- intercepts in a Lineweaver-Burk plot represent?

The x-intercept represents -1/Km and the y-intercept represents 1/Vmax.

thiamine & thiamine deficiency effects

Thiamine is an essential cofactor for several enzymes involved in cellular metabolism an nerve conduction. Thiamine deficiency (often a result of excess alcohol consumption and poor diet); results in diseases such as Wernicke-Korsakoff syndrome. Patients can suffer from neurologic deficits, including delirium, balance problems, and in severe cases, the inability to form new memories.

What are some examples of transient and covalent enzyme modifications?

Transient: allosteric activation or inhibition Covalent: phosphorylation & glycocosylation

fat-soluble vitamins

Vitamins A, D, E, and K -better regulated by partition coefficients

water-soluble vitamins

Vitamins B and C -B complex vitamins and ascorbic acid *must be replenished regularly because they are easily excreted

effect of salinity on enzyme activity

While the effect of salinity is not generally of physiologic significance, altering the concentration of salt can change enzyme activity in vitro. Increasing levels of salt can disrupt hydrogen and ionic bonds, causing a partial change in the conformation of the enzyme, and in some cases causes denaturation.

Why are some enzymes released as zymogens?

Zymogens are precursors of active enzymes. It is critical that certain enzymes (like digestive enzymes of the pancreas) remain inactive until arriving at their target site.

irreversible inhibition

active site is made unavailable for prolonged period of time or enzyme is permanently altered ex. aspirin

allosteric enzymes

alternate between active and inactive forms

uncompetitive inhibition

bind only to the enzyme-substrate complex and essentially lock the substrate in the enzyme, preventing its release. -Must bind at the allosteric site -lower Km and Vmax -On a Lineweaver-Burk plot, the curves for activity with and without the inhibitor are parallel.

allosteric inhibitior

binding will result in a shift that makes the active site less available for binding to the substrate

allosteric activator

binding will result in a shift that makes the active site more available for binding to the substrate

ligases

catalyze addition or synthesis reactions, generally between large similar molecules and often require ATP -likely to be encountered in nucleic acid synthesis and repair

oxidoreductases

catalyze oxidation-reduction reactions that involve the transfer of electrons reductant = electron donor oxidant = electron acceptor *enzymes with dehydrogenase and reductase

hydrolases

catalyze the breaking of a compound into two molecules using the addition of water *many hydrolases are named only for their substrate ex. phosphatase peptidase nuclease lipase

lyases

catalyze the cleavage of a single molecule into two products -do not require water as a substrate and do not act as oxidoreductases

transferase

catalyze the movement of a functional group from one molecule to another ex. kinases

isomerases

catalyze the rearrangement of bonds within a molecule -some can also be classified as oxidoreductases, transferases, or lyases depending on the mechanism of the enzyme

kinases

catalyze the transfer of a phosphate group, generally from ATP, to another molecule

synthase

catalyzes the synthesis of two molecules of a substance into a single molecule

phosphorylation/dephosphorylation

covalent modification of an enzyme -one cannot predict whether it will activate an enzyme without experimental determination

glycosylation

covalent modification of an enzyme -the covalent attachment of sugar moieties *can tag an enzyme for transport within the cell, or can modify protein activity and selectivitiy

catalysts

do not impact the thermodynamics of a biological reaction; that is, the ΔHrxn and equilibrium position do not change -help the reaction proceed at a much faster rate -not changed during the course of the reaction

Lineweaver-Burk Plot

double reciprocal graph of the Michaelis-Menten equation -yields a straight line x-axis = 1/Km y-axis = 1/vmax -useful when determining the type of inhibition that an enzyme is experiencing because Vmax and Km can be compared without estimation.

feedforward regulation

enzymes are regulated by intermediates that precede them

oxidase

enzymes in which oxygen is the final electron acceptor (oxidoreductase)

holoenzymes

enzymes with their cofactors

apoenzymes

enzymes without their cofactors

negative feedback

feedback inhibition; helps maintain homeostasis, once there is enough product, we turn off the pathway that creates the product.

cofactors

generally inorganic molecules or metal ions and are often ingested as dietary minerals

enzymes

important biological catalysts

zymogens

inactive form of an enzyme (precursor) -contain a catalytic domain and regulatory domain -the regulatory domain must be either removed or altered to expose the active site

enzyme-substrate (E-S) complex

interaction between substrate and enzyme

Kcat

kcat = Vmax/[E]t turnover #

active site

location within the enzyme where the substrate is held during the chemical reaction -assumes a defined spatial arrangement in the E-S complex, which dictates enzyme specificity -hydrogen bonding, ionic interactions, and transient covalent boding stabilize this spatial arrangement and contribute to the efficiency of the enzyme

induced fit model

more scientifically accepted model (over lock and key theory) -substrate induces a change in the shape of the enzyme -requires energy (endergonic)

Km (Michaelis constant)

often used to compare enzymes -measure of the affinity of the enzyme for its substrate Higher Km = lower affinity for substrate (requires higher substrate concentration to be half-saturated) Lower Km = higher affinity for substrate *Km is an intrinsic property of the E-S system and cannot be altered by changing concentration of substrate or enzyme.

exergonic reaction

one in which energy is given off (ΔG < 0)

endergonic reaction

one that requires energy input (ΔG > 0)

Hill's coefficient

quantifies cooperativity > 1: positive cooperative binding < 1: negative cooperative binding = 1: the enzyme does not exhibit cooperative binding

mixed inhibition

results when an inhibitor can bind to either the enzyme or the enzyme-substrate complex, but has different affinity for each -binding at an allosteric site -alters Km depending on the preference of the inhibitor for the enzyme vs. E-S complex --> free enzyme: Km increases E-S complex: Km decreases -On a Lineweaver-Burk plot, the curves for the activity with and without the inhibitor intersect at a point that is not on either axis.

coenzymes

small organic groups, the vast majority of which are vitamins or derivatives of vitamins such as NAD+, FAD, and coenzyme A

lock and key theory

suggest that the enzyme's active site (lock) is already in the appropriate conformation for the substrate (key) to bind -no alteration of the tertiary or quaternary structure is necessary upon binding of the substrate

saturation

the enzyme is working at maximum velocity and has reached Vmax (additional substrate has no effect) The only way to increase Vmax is to increase enzyme concentration by inducing expression of the gene encoding the enzyme.

substrate

the molecule upon which an enzyme acts

enzyme specificity

the molecules upon which an enzyme acts are called substrates; a given enzyme will only catalyze a single reaction or class of reactions with these substrates

feedback regulation

the regulation of the activity of an enzyme by one of its products

prosthetic groups

tightly bound cofactors or coenzymes necessary for enzyme function

Michaelis-Menten plot

-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. v = Vmax[S]/Km + [S] At 1/2 Vmax, Km = [S] (Graphs as hyperbola)

effects of local conditions on enzymes

-Temperature -Acidity or Alkalinity (pH) -High salinity all have significant effects on the ability of an enzyme to carry out its function

noncompetitive inhibition

-bind to an allosteric site instead of the active site, which induces a change in enzyme conformation -CANNOT be overcome by adding more substrate --> Km remains unaffected (same affinity) -Vmax is decreased (less enzyme available to react)

Key features of enzymes

-lower the activation energy -increase the rate of the reaction -do NOT alter the equilibrium constant -not changed or consumed in the reaction -are pH and temperature-sensitive, with optimal activity at specific pH ranges and temperatures -do not affect the overall ΔG of the reaction -are specific for a particular reaction or class of reactions

cofactors & coenzymes

-nonprotein molecules that participate in catalysis -tend to be small in size so they can bind to the active site and carry charge through ionization, deprotonation, or protonation -usually kept at low concentrations in the cell and only recruited when needed

competitive inhibition

-simply involves occupancy of the active site -can be overcome by adding more substrate so that the substrate to inhibitor ratio is higher -does NOT alter Vmax -increases Km (since more substrate is required to reach half of maximal velocity)

What are the four types of reversible inhibitors?

1) Competitive inhibitors 2) Noncompetitive inhibitors 3) Mixed inhibitors 4) Uncompetitive inhibitors

allosteric site

A site on an enzyme other than the active site, to which a specific substance binds, thereby changing the shape and activity of the enzyme.

Consider a reaction catalyzed by enzyme A with a Km value of 5 x 10^-6 M and vmax of 20 mmol/min. At a concentration of 5 x 10^-6 M substrate, the rate of the reaction will be: A) 10 mmol/min B) 20 mmol/min C) 30 mmol/min D) 40 mmol/min

A) 10 mmol/min

Which of the following statements about enzyme kinetics is FALSE? A) An increase in the substrate concentration (at constant enzyme concentration) leads to proportional increases in the rate of the reaction. B) Most enzymes operating in the human body work best at a temperature of 37 C. C) An enzyme-substrate complex can either from a product or dissociate back into the enzyme and substrate. D) Maximal activity of many human enzymes occurs around pH 7.4

A) An increase in the substrate concentration (at constant enzyme concentration) leads to proportional increases in the rate of the reaction. An increase in the substrate concentration, while maintaining a constant enzyme concentration, leads to proportional increase in the rate of the reaction only initially. However, once most of the active sites are occupied, the reaction rate levels off.

Which of the following factors determine an enzyme specificity? A) The three-dimensional shape of the active site B) The Michaelis constant C) The type of cofactor required for the enzyme to be active D) The prosthetic group on the enzyme

A) The three-dimensional shape of the active site

Which of the following is LEAST likely to be required for a series of metabolic reactions? A) Triacylglycerol acting as a coenzyme B) Oxidoreductase enzymes C) Magnesium acting as a cofactor D) Transferase enzymes

A) Triacylglycerol acting as a coenzyme Triglycerides are unlikely to act as coenzymes for a few reasons, including their large size, neutral charge, and ubiquity in cells.

Enzymes increase the rate of a reaction by: A) decreasing the activation energy B) decreasing the overall free energy change of the reaction C) increasing the activation energy D) increasing the overall free energy change of the reaction

A) decreasing the activation energy

What are the effects of temperature, pH, and salinity on the functions of enzymes?

As temperature increases, enzyme activity generally increases (doubles every 10 C). Above body temperature, enzyme activity quickly drops off as the enzyme denatures. Enzymes are maximally active within a small pH range; outside of this range, activity drops quickly with changes in pH as the ionization of the active site changes and the protein is denatured. Changes in salinity can disrupt bonds within an enzyme, causing disruption of tertiary and quaternary structure, which lead to loss of enzyme function.

How does the ideal temperature for a reaction change with and without an enzyme catalyst? A) The ideal temperature is generally higher with a catalyst than without B) The ideal temperature is generally lower with a catalyst than without. C) The ideal temperature is characteristic of the reaction, not the enzyme. D) No conclusion can be made without knowing the enzyme type.

B) The ideal temperature is generally lower with a catalyst than without.

Some enzymes require the presence of a nonprotein molecule to behave catalytically. An enzyme devoid of this molecule is called a(n): A) holoenzyme B) apoenzyme C) coenzyme D) zymoenzyme

B) apoenzyme

B vitamins

B1: thiamine B2: riboflavin B3: niacin B5: pantothenic acid B6: pyridoxal phosphate B7: biotin B9: folic acid B12: cyanocobalamin

How are the Michaelis-Menten and Lineweaver-Burk plots similar? How are they different?

Both the Michaelis-Menten and Lineweaver-Burk relationships account for the values of Km and Vmax under various conditions. They both provide simple graphical interpretations of these two variables and are derived from the Michaelis-Menten equation. However, the axes of these graphs and the visual representation of this information differs between the two. Michaelis-Menten plot is v vs. [S], which creates a hyperbolic curve for monomeric enzymes. The Lineweaver-Burk plot, is 1/v vs. 1/[S] and creates a straight line.

Consider a reaction catalyzed by enzyme A with a Km value of 5 x 10^-6 M and vmax of 20 mmol/min. At a concentration of 5 x 10^-4 M substrate, the rate of the reaction will be: A) 10 mmol/min B) 15 mmol/min C) 20 mmol/min D) 30 mmol/min

C) 20 mmol/min At a concentration of 5 x 10^-4 M, there is 100 times more substrate than present at half maximal velocity. At high values, the enzyme is at or near Vmax, which is 20 mmol/min.

The conversion from ATP to cyclic AMP and inorganic phosphate is most likely catalyzed by which class of enzyme? A) Ligase B) Hydrolase C) Lyase D) Transferase

C) Lyase Lyases are responsible for the breakdown of a single molecule into two molecules without the addition of water or the transfer of electrons. Lyases often form cyclic compounds or double bonds in the products to accommodate this.

How do enzymes function as biological catalysts?

Catalysts are characterized by two main properties: they reduce the activation energy of a reaction, thus speeding up the reaction, and they are not used up in the course of the reaction. Enzymes improve the environment in which a particular reaction takes place, which lowers its activation energy. They are also regenerated at the end of the reaction to their original form.