Mechanisms and Inhibitors

Question 1

*what environmental factors can affect enzyme activity?

Answer 1

Temperature, pH and inhibitory molecules

Question 2

*Explain the difference between reversal and irreversible inhibition of enzyme activity.
What kind of binding does Irreversible inhibition create to elucidate mechanisms for enzymes?

Answer 2

Reversal inhibition: bind to active site, and RAPIDLY dissociate from enzyme-substrate complex
Irreversible- inhibitors binds very tightly to an enzyme. (mainly covalently). they bind to a group at active site, dissociate SLOWLY from Target enzyme inhibitors that bind enzyme covalently, are powerful for elucidating mechanisms of enzyme action

Question 3

*Write the enzyme, substance reaction sequence for competitive inhibition? Draw what the graphs of velocity versus substrate look like.

Answer 3

competitive inhibition: inhibitor is structurally similar to substrate and binds to active site of enzyme; prevents substrate from binding. Substrate and inhibitor bind SAME site.
Increasing substrate concentration will outcompete inhibitor and overcome inhibition.
Km app INCREASES (more substrate), Vmax stays the same (UNCHANGED).
substrate/inhibitor ratio
Km app (apparent value of Km) increases w/ inhibitor

Question 4

*Write the enzyme, substance reaction sequence for un-competitive inhibition? Draw what the graphs of velocity versus substrate look like.

Answer 4

Uncompetitive inhibition- inhibitor binds only to Enzyme-Substrate Complex. Inhibitor can only bind after the substrate binds.
Km DECREASES, Vmax DECREASES
inhibitor bind to ES complex forming ESI which will make not form any product. due to unproductive ESI complex present, V max will be lowered when inhibitor is included. Also, Km decreases since ESI diminishes ES complex. Km small due to affinity for substrate increasing.
Uncompetitive inhibition CANNOT be overcome by adding excess substrate
Substrate dependent

Question 5

*Write the enzyme, substance reaction sequence for non-competitive inhibition? Draw what the graphs of velocity versus substrate look like.

Answer 5

Non-competitive inhibition- inhibitor can bind either the free enzyme or enzyme-substrate complex.
Either case, the binding prevents formation of product.
Binding of inhibitor is independent of binding of substrate.
Vmax DECREASES, Km UNCHANGED
Vmax is lower in presence of non-competitive inhibitor, and km unchanged.
Noncompetitive inhibition CANNOT be overcome by adding excess substrate.

Question 6

What are the different kinds of catalytic strategies?

Answer 6

1. covalent catalysis- active site contains nucleophile that briefly modified ex: chymotrypsin
2. General acid-base- a molecule other than water donates or accepts a proton (Histidine)
3. Metal Ion catalysis- metal ions function as electrophilic catalyst (stabilize neg charge on intermediate), other functions (generate nucleophile by increasing acidity of H2O, bind substrate, increasing interactions with enzyme)
4. Catalysis by approximation and orientation: enzyme brings 2 substrates together in an orientation (of single binding surface of enzyme) that facilitates catalysis.

Question 7

What is a nucleophile? What is an electrophile?

Answer 7

Nucleophile- chemical that is attracted to regions of positive charge, and involved in chemical reactions, donate e- to electrophiles.
Electrophile- chemical that is electron deficient, and are attracted nucleophiles (e- rich molecules)

Question 8

What is a major key aspect of enzyme catalysis?

Answer 8

Binding energy- interactions between substrate and enzyme that is formed only when substrate is in transition state.

Question 9

how does temperature affect enzyme activity? What occurs when the temperature is too high? How does it affect Brownian motion? What eventually happens to enzyme activity at a certain temp?

Answer 9

Temperature enhances enzyme activity. Higher the temperature, the higher the rate of reaction (faster reaction occurs).
increase in temperature leads to increase in Brownian motion and makes interactions between substrate and enzyme more likely.
increase in temperature will also increase enzyme activity, until the enzyme denatures. At a certain temperature, heat will disrupt weak forces between 3D structure of a protein, causing denaturation.

Question 10

How does temp affect humans (endotherms) versus reptiles (ectotherms)

Answer 10

Humans regulate certain body temperature and ambient (outside temps) do not really minimize enzyme activity. However reptiles like lizards, use temperatures as important regulators of important biochemical and biological activity. ex: lizards are more active in hotter temperatures, and inactive in cooler temps.

Question 11

which organisms can live in temperatures that would normally denature proteins?

Answer 11

Archaea- they live in hot environments with temps over 80 degrees Celsius, near volcanic vents. They are very resistant to thermal denaturation

Question 12

How does pH affect enzyme activity? provide 2 main examples.

Answer 12

Most enzymes have optimal pH- the pH at which enzymes display maximal activity.
Optimal pH varies with each enzyme and is correlated to its specific environment.
Ex: Pepsin -protein digesting enzyme functions in highly ACIDIC environments in STOMACH at pH of 1 or 2 (pH level where most proteins denatured)
Chymotrypsin- have optima pH of 7 or 8 and function in pancreas, upper site of small intestine

Question 13

What factor contributes to an enzyme’s dependence on pH? What’s an example?

Answer 13

Due to presence of ionizable R groups.
Ex: if functional enzyme requires ionization of lysine (positively charged) and glutamic acid (negatively charged), enzmye would depend on presence of COO- and NH3+ group.
if pH is lowered, more H+ [ } will be gained, and COO- will convert to COOH causing loss of enzyme activity. if pH is raised (H+ will go to OH- (more of this gained) and NH3+ will lose an H+ to become NH2 (neutral ) and will diminish enzyme activity.

Question 14

What are the three common types of reversible inhibition? How do they differ?

Answer 14

competitive, non-competitive and un-competitive inhibition.

These three differ by the interaction between inhibitor and enzyme and inhibitor’s effect on enzyme kinetics.

Question 15

What occurs when there is more substrate concentration in competitive inhibition?

Answer 15

High substrate concentration will allow all active sites to be occupied by substrates and enzyme will be fully operative
More inhibitors present, more substrate needed to displace them and reach vmax

Question 16

What are examples of inhibitors (drugs) for each reversible inhibitor?

Answer 16

Competitive- Sulfanialmide (sulfa drug) is  a class of antibiotics contain Sulfur atom, and is structurally similar to PABA (p-aminobenzoic acid) which is a substrate used to synthesize folic acid. inhibitor binds to active site and block PABA (hence no synthesis of folic acid); also statin and ibroprufen
Uncompetitive- herbicide Roundup inhibits synthesis of amino acids in plants, causing plants to die. 
Non-competitive- Doxycycline- antibiotic that functions at low [  ] inhibits collagenase (bacterial proteolytic enzyme) and prevent growth and reproduction of bacteria in gum disease.

Question 17

what kind of graph highlights differences in types of reversal inhibition? Describes shape of graphs for each.

Answer 17

DOUBLE RECIPROCAL plots (LINE-weaver burker)
competitive- slope increases, y-intercept same with or without inhibitor (x shape)
uncompetitive- slope stays same, y intercept increased with inhibitor (parallel lines)
non-competitive - slope and y-intercept increase (V shape)

Question 18

What are the four categories of irreversible inhibitors?

Answer 18

Group specific reagents, affinity labels, suicide inhibitors and transition state inhibitors.

Question 19

How do group specific reagents function? Provide an example

Answer 19

group specific reagents react with R groups of specific amino acids.
Ex: reagent DIPF (diisopropylphosflouridate) inactivates enzyme acetylcholinesterase (important in transmitting nerve impulses) by modifying reactive serine group and binding to inactivated enzyme.
DIPF (potent nerve gas) also inhibits chymotrypsin by modifying one serine residue out of 28 serine groups in enzyme.

Question 20

How do suicide inhibitors function?

Answer 20

Suicide inhibitors or mechanism based inhibitors bind to enzyme as a substrate. As catalysis occurs, enzyme modifies substrate converting it into inhibitor (irreversibly inactivating enzyme)
Ex: Penicillin is a suicide inhibitor of enzyme that synthesizes bacterial cell walls.

Question 21

How do transition state inhibitors work? Affinity labels?

Answer 21

They closely resemble the transition state of substrate (bind and inhibit enzyme)
affinity labels- covalently modify active site residues and are structurally similar to substrate (ex TPCK binds to active site, modify His residue, inactivate chymotrypsin).

Question 22

Explain the role of Penicillin as an inhibitor? Describe the structure of penicillin and how it inhibits an enzyme.

Answer 22

Penicillin is a SUICIDE INHIBITOR.
structure: thiazolidine ring fused to reactive Beta lactam ring, and R group
Penicillin inhibits the formation of cell walls in Bacterial S. aureus. Cell wall of S. aureus is made from molecule peptidoglycan which is a linear polysaccharide chain cross-linked by short peptides.
Glycopeptide transpeptidase - enzyme catalyzes peptide-cross links, and the transpeptidase reaction proceeds to acyl-enzyme intermediate. \
Penicillin binds to peptidase, a serine residue will attack the carbonyl carbon of lactam ring, as if penicillin were a substrate
A pencinillonyl-serine derivative is then formed, and is enzymatically inactive and very stable