Enzymes

Isomerases

Catalyze the interconversion of isomers, including both constitutional isomers and stereoisomers.

Glycosylation

Covalent modification with carbohydrate, can alter the activity or selectivity of enzymes.

Phosphorylation

Covalent modification with phosphate, can alter the activity or selectivity of enzymes.

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

D. Breaking bonds in the enzyme irreversibly to provide energy *All other statements are correct

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 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. * As each subunit binds to the substrate, affinity is greater after each subunit binds. This is true for dissociating as well, which is why "A" is not a strong statement because the unit has affinity for both association and dissociation, so the statement is not specific enough to be correct.

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 change (ΔG) of the catalyzed reaction is the same as for the uncatalyzed reaction.

D. The free energy change (ΔG) of the catalyzed reaction is the same as for the uncatalyzed reaction.

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 --enzyme 1-->B--enzyme 2--> C A. competitive inhibition B. irreversible ihibition C. feedback inhibition D. negative feedback

D. negative feedback *When the product (substrate C) in an equation is inhibitory to an earlier part of the equation (due to high levels of product) the process is called negative feedback. This mechanism helps conserve energy by stopping production when product is at high levels.

Michaelis-Menten Equation

Describes how the rate of the reaction "v", depends on the concentration of both the enzyme, [E], and the substrate, [S], which forms product, [P]. Enzyme-substrate complexes form at rate k1. The ES complex can either dissociate at a rate k2 or turn into E + P at rate k3: E + S ⇌ ES ➝ E + P v =Vmax[S]/Km+[S]

Thermodynamics vs. Kinetics

Enzymes do NOT alter the free energy (ΔG) or enthalpy (ΔH) change that accompanies the reaction nor the final equilibrium position ; rather they change the rate (kinetics) at which equilibrium is reached.

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

Enzymes have no affect on the overall thermodynamics of the reaction; they have no affect on the 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 the equilibrium position does not change.

Cofactors

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

Allosteric Enzymes

Have multiple binding sites, active site is presents and at least one other site can regulate the availability of the active site. Alternate between an active and inactive form. Molecules that bind to the allosteric site may be activators or inhibitors, and binding causes a conformational change. Binding of allosteric activator or inhibitor may alter the activity of enzymes.

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 [S] is low, an increase in [S] causes a proportional increase in enzyme activity. At high [S] , however, when the enzyme is saturated, increasing [S] has no effect on activity because Vmax has already been attained. Increasing [E] will always increase Vmax, regardless of the starting [E].

Feedback Inhibition

Is a regulatory mechanism whereby catalytic activity of an enzyme is inhibited by the presence of high levels of a product later in the same pathway.

Reversible Inhibition

Is characterized by the ability to replace the inhibitor with a compound of greater affinity or to remove it using mild laboratory treatment.

1/2 Vmax

Km = [S] Km can therefore be understood to be the substrate concentration at which half of the enzyme's active sites are full. Km is the Michaelis constant, often used to compare enzymes.

How do the lock and key and induced model differ?

Lock and key: active site of enzyme fits exactly around the substrate. No alterations to tertiary and quaternary structure of the enzyme. Less accurate model. Induced fit: Active site of the enzyme molds itself around substrate only when substrate is present. Tertiary and quaternary structure is modified for enzyme function. More accurate model.

What is enzyme 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.

Uncompetitive Inhibition

Results when the inhibitor binds only with the enzyme- substrate complex. Because the substrate- enzyme complex has already formed, the inhibitor must bind to an allosteric site on the complex. Km and Vmax both decrease.

Noncompetitive Inhibition

Results when the inhibitor binds with equal affinity to the enzyme and the enzyme-substrate complex. The inhibitor binds to an allosteric site instead of the active site, which induces an enzyme conformation. Because the two molecules do not bind to the same site, they are considered noncompetitive; and also cannot be overcome increasing substrate due to the conformation change. Vmax is decreased because there is less enzyme there to react, Km is unchanged because affinity stays the same for existing enzyme.

Mixed Inhibition

Results when the inhibitor binds with unequal affinity to the enzyme and the enzyme- substrate complex. Vmax is decreased, Km is increased or decreased depending on if the inhibitor has higher affinity for the enzyme or enzyme- substrate complex.

Competitive Inhibition

Results when the inhibitor is similar to the substrate and binds at the active site. Competitive inhibition can be overcome by adding more substrate. Vmax is unchanged, Km increases.

Coenzymes

Small organic groups, the vast majority of which are vitamins or derivatives of vitamins such as NAD+, FAD, and coenzyme A. The water- soluble vitamins include the B complex vitamins and ascorbic acid (vitamin C).

Lock and Key Theory

Suggests that 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. No alteration of the tertiary and quaternary structure is necessary upon binding of the substance.

Effects of Local Conditions on Enzyme Activity

Temperature and pH affect an enzyme's activity in vivo; changes in temperature and pH can result in denaturing of the enzyme and loss of activity due to loss of secondary, tertiary, and quaternary structure. In vitro, salinity can impact the action of enzymes.

Enzyme specificity

The fact that each enzyme catalyzes a single reaction or type of reaction with high specificity.

Induced Fit Model

The more scientifically accepted theory, starts with a substrate and enzyme active site that don't seem to fit together. However, once the substrate is present and ready to interact with the active site, the molecules find that the induced form, or 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.

Saturation

The point at which all enzyme is bound to substrate and the rate of the reaction can not be increased. At this rate the enzyme is working at maximum velocity, denoted my Vmax.

Km

When comparing two enzymes, the enzyme with the higher Km has the lower affinity for its substrate because it requires a higher substrate concentration to be half saturated. The Km is an intrinsic property of the enzyme and cannot be altered by changing the concentration of substrate or enzyme.

Ligases

are responsible for joining two large biomolecules, often of the same type.

Hydrolases

catalyze cleavage with the addition of water.

Cooperative enzymes

display a sigmoidal curve because of the change in activity with substrate binding

Transferases

move a functional group from one molecule to another molecule.

Exergonic Reaction

releases energy and ΔG is negative

Lineweaver- Burk Plots

A double reciprocal graph of the Michaelis- Menten equation. The same data graphed in this way yield a straight line. The actual data are represented by the portion of the graph to the right of the y-axis, but the line is extrapolated into the upper left quadrant to determine its intercept with the x-axis. The intercept of the line with the x-axis gives the value of 1/ Vmax. Useful when determining the type of inhibition that an enzyme is experiencing because Vmax and Km can be compared without estimation.

The activity of an enzyme is measured at several different substrate concentrations, and the data are shown below. [S] (mM) (v [mmol/sec] ) 0.01 1 0.05 90 0.1 17 0.5 50 1 67 5 91 10 95 50 99 100 100 Km for this enzyme is approximately: A. 0.5 B. 1 C. 10 D. 100

A. 0.5 *Km is when the enzyme is filled half with substrate.

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 * If Vmax is 20 mmol/ min (full saturation of enzyme, then Km is 1/2 of Vmax, when the enzyme is at half saturation, which would be 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 37°C. C. An enzyme-substrate complex can either form 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. *Saturation makes this statement false.

Which of the following factors determine an enzyme's specificity? A. The 3D 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 3D shape of the active site

Which of the following is LEAST likely to be required for 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 *Least likely because of 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

Maximum Velocity

Achieved when all enzyme is bound to substrate also known as saturation. The only way to increase Vmax is to increase enzyme concentration. In a cell this can be induced by inducing the expression of the gene encoding the enzyme.

Enzyme function

Act by stabilizing the transition state, providing a favorable microenvironment, or bonding with the substrate molecules. Active site is the site of catalysis. Some require metal cation cofactors or small organic coenzymes to be active. Enzymatic reactions are NOT restricted to a single cofactor or coenzyme. For example metabolic reactions often require magnesium, NAD+ (derived from vitamin B), and biotin (B7) simultaneously.

Irreversible Inhibition

Alters the enzyme in such a way that the active site is unavailable for prolonged duration or permanently; new enzyme molecules must be synthesized for the reaction to occur again.

Enzymes

Are biological catalysts that are unchanged by the reactions they catalyze and are reusable.

Zymogen

Are secreted in an inactive form and are activated by cleavage.

Saturation Kinetics

As substrate concentration increases the reaction rate does as well until a maximum value is reached.

The graph below shows kinetic data obtained for flu virus enzyme activity as a function of substrate concentration in the presence and absence of two antiviral drugs. Based on the graph, which of the following statements is correct? A. Both drugs are noncompetitive inhibitors of the viral enzyme. B. Tamiflu increases the Km value for the substrate compared to Relenza. C. Relenza increases the Vmax value for the substrate compared to Tamiflu D. Both drugs are competitive inhibitors of the viral enzyme.

B. Tamiflu increases the Km value for the substrate compared to Relenza. *When comparing the lines plotted, remember that the Y-int = 1/Vmax and the X-int = -1/ Km. So we see that Tamiflu and no inhibitor lines have the same Vmax and Tamiflu has a larger Km, which is characteristic of Competitive inhibition. Relenza has the same Km as the no inhibitor line and a decreased Vmax, which is characteristic of noncompetitive.

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. *enzymes tend to denature at high temperatures, so the presence of an enzyme will lower the ideal temperature.

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

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 *The new [S] is 100 x higher than Km, at these levels the rate will be at or near Vmax, 20 mmol/min.

The conversion of 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

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.

Lyases

Catalyze cleavage without the addition of water and without the transfer of electrons. The reverse reaction (synthesis) is often more important biologically.

Oxidoreductase

Catalyze oxidation- reduction reactions that involve the transfer of electrons.