MMT: Catalysis I and II

Question 1

state how enzymes speed up the rates of reactions

Answer 1

by lowering the activation energy required to reach the transition state

Question 2

how does an enzyme impact Keq and free energy

Answer 2

it does not; the only thing impacted is the activation energy and therefore the rate of the reaction

Question 3

describe the basic structures and properties of enzymes

Answer 3

enzymes are proteins with highly specific binding sites for the substrates they bind; some contain multiple catalytic sites. in general, substrate binds to the active site and convert to product. they also have allosteric sites (sites other than the active site) that modifiers can bind to in order to regulate enzymatic activity. and some require cofactors.

Question 4

Differentiate between lock-and-key and induced-fit models of substrate binding

Answer 4

1. lock and key: the shape of the active site is complimentary to the shape of the substrate
2. induced fit: the shape of the active site changes upon substrate binding to become complementary to the shape of the susbtrate

Question 5

what is the primary way in which interactions between substrate and enzyme are stabilized?

Answer 5

non-covalent interactions such as H-bonding, ionic bonding, and hydrophobic interactions

Question 6

Explain the process and purpose of substrate tunneling or channeling in multifunctional enzymes. name an example enzyme.

Answer 6

process: some enzymes have multiple catalytic sites, allowing them to catalyze more than one reaction. one reaction takes place, and the product is shunted to another nearby catalytic site on the same enzyme for a subsequent reaction.

purpose: increased efficiency and reaction rate, prevents loss of intermediates

big ex. is carbamoyl phosphate synthetase!

Question 7

Specify some factors that may affect enzyme activity, and explain how they may do so.

Answer 7

pH: can influence ionization states of sidechains of amino acids that participate in ionic interactions. non-optimal pH can denature the enzyme and/or impact their ability to catalyze reactions

temperature: high temperatures can denature enzymes and prevent them from functioning

Question 8

List the six categories of enzymes and briefly describe the reactions they catalyze

Answer 8

1. oxidoreductases: oxidation-reduction; transferring electrons
2. transferases: transferring groups
3. hydrolases: hydrolysis reactions i.e. using water to break bonds
4. lyases: removing or adding groups to form double bonds
5. isomerases: isomerization
6. ligases: join large molecules at the expense of ATP hydrolysis. require lots of energy!

Question 9

Define cofactor, coenzyme, and prosthetic group.

Answer 9

cofactor: small molecules that perform chemical reactions that the enzyme itself is unable to do. they can be small metal ions or coenzymes

coenzyme: a subcategory of cofactors that are small organic molecules. they tend to be derived from vitamins, often a b-complex vitamin.

prosthetic group: a tightly bound coenzyme

Question 10

Explain how initial velocity of a reaction is determined.

Answer 10

enzymatic assays are carried out, measuring the differences in formation of product with various substrate concentrations. the slope of the reaction curve is then taken to find V0

Question 11

Define the terms Km and Vmax and explain how you would determine them

Answer 11

Vmax: the maximum possible reaction speed; determined by the velocity observed when the addition of more substrate does not impact the reaction speed

Km: a constant for any given enzyme; the substrate concentration at 1/2 of the maximum velocity. it shows the relative affinity of substrate to enzyme; a higher Km means a lower affinity!

Question 12

Explain the difference between a Michaelis-Menten plot and a Lineweaver-Burk plot in
analyzing enzyme kinetics

Answer 12

it is easier to determine exact Km and Vmax values from a lineweaver-burk plot. a lineweaver-burk plot has 1/[s] as the x axis, and 1/V as the y. this makes the x intercept 1/Km, the y intercept 1/Vmax, and the slope Km/Vmax. the MM equation plots [s] on the x axis and velocity on the y, making it less specific in terms of finding Vmax and Km.

Question 13

Determine the Km and Vmax values from a Lineweaver-Burk plot

Answer 13

Vmax: look at the Y intercept; a higher-up intercept means a lower Vmax

Km: look at the X intercept; a left-shifted intercept means a lower Km

Question 14

define irreversible and reversible inhibition

Answer 14

irreversible: the inhibitor bonds covalently to the enzyme, making it permanently inactive. the only way to break the bond is to degrade the whole protein

reversible: the inhibitor binds non-covalently, and can dissociate from the enzyme. examples are competitive, uncompetitive, and non-competitive inhibition.

Question 15

define a suicide inhibitor

Answer 15

a type of irreversible inhibitor where the molecule partially reacts with the active site to form a covalently linked intermediate product. examples are penicillin and 5FU

Question 16

Describe the underlying mechanisms of competitive inhibition and give examples

Answer 16

the inhibitor is structurally similar to the substrate, and competes with the substrate to bind to the active site. relative concentration and affinity impact the degree of competition.

ex:
- methotrexate mimics dihydrofolate and inhibits nucleotide synthesis in cancer cells
- statins mimic HMG-CoA and inhibit cholesterol synthesis
- ethanol inhibits the binding of methanol and formation of formic acid, and is used to treat methanol poisoning

Question 17

Describe the underlying mechanisms of non-competitive inhibition and give
examples

Answer 17

an inhibitor binds to an allosteric site, either on the enzyme or the ES complex, and changes the conformation. adding more substrate cannot overcome this.

ex:
- acetazolamide is an inhibitor of carbonic anhydrase and is used as a diuretic

Question 18

Specify how competitive, non-competitive and un-competitive inhibitors affect the Km and
Vmax of a reaction.

Answer 18

competitive: same Vmax, increased Km. the inhibitor binds to the active site but doesn’t react. by adding more substrate, the inhibitor can be displaced. this is why Km is increased, as it takes more substrate to be able to reach 1/2 Vmax

non-competitive: decreased Vmax, same Km. no matter how much substrate is added, enzymes are taken out of commission so overall Vmax is lowered.

un-competitive: Km and Vmax are decreased. inhibitor binds to ES complex

Question 19

Describe the difference in velocity vs. substrate concentration (Michaelis-Menten) curves for
regulatory and non-regulatory enzymes

Answer 19

regulatory enzymes tend to display a sigmoidal-shape curve

Question 20

Define the terms cooperativity and allostery.

Answer 20

cooperativity: process by which the binding of a substrate at one site of an enzyme impacts the binding of substrate at other sites. cooperativity can be positive or negative.

allostery: process by which the binding of molecules at allosteric sites impacts the binding of/affinity for susbtrate. allostery can be positive or negative

Question 21

Explain the effects of negative and positive modifiers on the Km of a regulatory enzyme

Answer 21

a positive modifier decreases Km, shifting the MM curve to the left. a negative modifier will increase Km, shifting the MM curve to the right.

Question 22

Describe the Bohr effect, with respect to hemoglobin, and its importance at the tissue and lung
levels

Answer 22

elevated H+ (aka lower pH) and increased CO2 levels decrease hemoglobins affinity for oxygen, allowing more oxygen to be released to the tissues. they decrease Km and shift the curve to the right, making them negative allosteric modifiers. in the lungs, a higher partial pressure of O2 will increase hemoglobin’s affinity for O2, causing it to bind (positive cooperativity).

Question 23

List three mechanisms of enzyme regulation in addition to allosteric modulation

Answer 23

1. isoenzymes
2. reversible covalent modifications
3. irreversible proteolytic activation

Question 24

describe regulation via reversible covalent modifications

Answer 24

a donor molecule transfers a functional group to a side chain on a target protein. this can impact enzymatic activity. a big example is phosphorylation/dephosphorylation via kinases/phosphatases

Question 25

describe regulation via isoenzymes

Answer 25

homologous enzymes with different but similar amino acid sequences. they have different kinetic parameters, and prevent fine-tuning of metabolism. ex is LDH, which has an M form in skeletal muscle and H form in the heart.

Question 26

describe regulation via irreversible proteolytic activation

Answer 26

zymogens (inactive precursers) are cleaved in order to become active; this process can only occur once and is irreversible. the zymogen is stored until it is ready for transport to be used. an example is chymotrypsinogen converting to chymotrypsin