Lecture 6 - Enzyme Mechanism & Regulation

Question 1

What are the 6 major classes of enzymes?

Answer 1

1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases

Question 2

What is a transferase?

Answer 2

Transfers functional groups from one substrate to another

like one that would do a nucleophilic substitution

Question 3

What is a hydrolase?

Answer 3

Enzyme which catalyzes a hydrolysis reaction, cleaving the substrate with the addition of water (OH to one, H to the other)

Question 4

What is a lyase?

Answer 4

Enzyme which catalyzes a lysis reaction without the use of water

Question 5

What is an isomerase?

Answer 5

Enzymes catalyzing isomerization reactions by rearranging atoms in a molecule

Question 6

What is a ligase?

Answer 6

Enzymes using ATP to ligate two molecules together. I.e. DNA ligase

Question 7

What are the two models for the enzyme substrate complex?

Answer 7

1. Lock and key model - substrate fits the active site perfectly
2. Induced fit model - active site fits the transition state to the product perfectly

Question 8

What are orientation and proximity effects?

Answer 8

Substrates are bound and held in optimum orientation for reaction to occur

Question 9

How can substrate strain play a part in enzyme activity?

Answer 9

Enzyme imposes strain or distortion on the substrate molecule that makes it easy to cleave the bond

Question 10

What is the passive vs active role of metal ion catalysis?

Answer 10

1. Passive role: Attract / neutralize negatively substrate material. For instance, Mg+2 is required for ATP to band an enzyme active site.
2. Active role: Increase the reactivity of groups involved in catalysis. For instance, Zn+2 coordinate of water and side chain atoms in the active site of carboxypeptidase A.

Question 11

What is meant by covalent catalysis?

Answer 11

Forming and breaking covalent bonds to the enzyme in order to catalyze a reaction. For instance, in chymotrypsin, Enzymatic Serine-OH group is a better nucleophile than water, and greatly speeds up proteolysis.

Question 12

What is a common example of protein denaturation?

Answer 12

Denaturation of albumen in egg whites, which causes cysteine to form disulfide bond crosslinks.

Question 13

How does substrate concentration differ in vivo vs in vitro?

Answer 13

In vivo: [S] is about equal to Km

In vitro: [S] is about 30-50 fold greater than Km to assess Vmax

Question 14

What is the Lineweaver-Burk plot and how is it derived?

Answer 14

Double-reciprocal plot, derived by taking the reciprocal of the Michaelis Menten formula.

Y-intercept is: 1/Vmax
Slope is Km/Vmax
X-intercept is: -1/Km

Question 15

What are the three types of reversible enzyme inhibition?

Answer 15

1. Competitive
2. Noncompetitive
3. Uncompetitive

Question 16

How are the Km apparent and Vmax changed in competitive inhibition? How does this appear in Lineweaver Burk?

Answer 16

Km increases - Relatively more substrate is needed to reach half Vmax, since inhibitor is sometimes binding

Vmax stays the same - you can add enough substrate to make Enzyme-Inhibitor Complex formation unlikely

Y intercept (1/Vmax) does not change
X intercept (-1/Km) moves more towards zero as [I] increases

Question 17

What is non-competitive inhibition and how does the affect the apparent Vmax and Km?

Answer 17

Inhibitor binds allosteric site, does not interfere with substrate binding, but slows enzyme. This happens equally well with free enzyme or ES complex.

Vmax decreases - enzyme cannot work as fast due to presence of [ESI] complex

Km is unchanged - competitive and uncompetitive effects of a noncompetitive inhibitor are equal. Any increase in Km from the binding of the inhibitor to free enzyme will be compensated by a decrease in Km from the uncompetitive stabilization of the ESI complex

Question 18

Why is it called noncompetitive?

Answer 18

Noncompetitive inhibitors do not bind at the active site, and are not competing for the same site. Thus, increasing [S] will not overcome inhibition, since they are not competing for binding. As long as inhibitor is present, true Vmax will never be achieved since [ES] will always be less than [E]subT (some will be ESI)

Question 19

How do irreversible inhibitors work?

Answer 19

They covalently bond the free enzyme, taking that enzyme out of action

Question 20

What kinetic profiles do irreversible inhibitors have and why?

Answer 20

Same as non-competitive inhibitors, because [ES] will always be less than [E]subT, because some enzymes will be [EI] due to irreversible binding to inhibitor. However, remaining enzymes have the same Km (same [S] needed to reach their lowered Vmax)

Question 21

What is uncompetitive inhibition and how does it influence Vmax and Km?

Answer 21

The inhibitor binds to ES complex and stabilizes it. It’s “uncompetitive” because it actually works synergistically with substrate binding, and helps lower Km

Vmax is decreased since [ES] < [E]subT, and some enzyme is tied up as ESI.

Km is lowered by making is harder for ES to dissociate, since it stabilizes it. Initial binding is just as easy, but once bound, it’s less likely to go back to E+S, and just says ESI.