Protein function and regulation

Question 1

What is the definition of a ligand?

Answer 1

The molecule to which a protein binds.

Question 2

What is the importance of specificity in ligand-binding?

Answer 2

It allows a protein to bind only one particular ligand, even in the presence of a vast excess of irrelevant molecules.

Question 3

What is affinity in ligand binding?

Answer 3

The tightness, or strength of binding, expressed as
dissociation constant (Kd).

Question 4

What is the relationship between the strength of
interaction and Kd?

Answer 4

The stronger the interaction, the lower the Kd.

Question 5

How does protein binding specificity arise?

Answer 5

It arises from numerous interactions which are individually weak, but, if numerous, collectively strong.

Question 6

What is binding in molecular interactions?

Answer 6

It is an interaction between complementary molecular surfaces.

Question 7

What are antibodies? (physical)

Answer 7

Antibodies contain two (identical) heavy chains and
two (identical) light chains.

Question 8

What is the antigen-binding surface of antibodies?

Answer 8

The antigen-binding surface or CDR (complementarity determining region) involves multiple protein loops from both the heavy and the light chains.

Question 9

What are enzymes?

Answer 9

Enzymes are an extremely diverse class of catalytically active proteins whose ligands include the substrates of the reactions they catalyze.

Question 10

What is the active site of an enzyme?

Answer 10

Substrate binding, and reaction catalysis, occur at the enzyme’s active site.

Question 11

Where does substrate specificity arise from?

Answer 11

Substrate specificity arises from the substrate binding site

Question 12

What is Vmax?

Answer 12

Vmax is the maximal rate of catalysis given saturating amounts of substrate.

Question 13

What is the turnover number?

Answer 13

The enzymatic cycles per second at top speed.

Question 14

What is Km?

Answer 14

Km is the substrate concentration that supports a rate of catalysis equal to one-half of the Vmax.

Question 15

What does Km depend on, is a measure of?

Answer 15

the affinity of enzyme:substrate binding

Question 16

4 times as much enzyme in the reaction gives a Vmax ________________, but the measured Km is the _______ because, in general, the binding affinity is __________ of concentrations, but depends only on the _________ properties of the enzyme and the substrate.

Answer 16

4‐fold higher
same
independent
chemical

Question 17

What do proteases do?

Answer 17

hydrolyze peptide bonds in polypeptides

Question 18

What are the serine proteases?

Answer 18

The serine proteases are a family of proteases whose catalytic mechanism involves a serine residue in the catalytic site.

Question 19

What is the mechanism of action of trypsin?
(hydrolyzes what? and what are the 2 steps?)

Answer 19

Trypsin hydrolyzes peptide bonds adjacent to arginine and lysine (large, basic side chains).

Step 1 : cleavage of peptide bond with formation of a covalent substrate-enzyme complex (Ser195 acyl enzyme).
Step 2 : hydrolysis of acyl enzyme complex

Question 20

What amino acid side chains are involved in the trypsin mechanism?

Answer 20

catalytic mechanism involving 3 amino acid side chains Asp-102, His-57, and Ser-195.

Question 21

What is the role of the negatively charged pocket in
the substrate binding site of trypsin?

Answer 21

Proper substrate binding only occurs when the substrate amino acid side chain “fits” into a negatively charged pocket within the substrate binding site.

Question 22

Differences in the substrate recognition pocket in the related enzymes define their differing ___________.

Answer 22

specificities

Question 23

What is the pH range at which trypsin subreactions reactions happen best?

Answer 23

depend on His‐57’s ability to bind, and to release a proton.
These reactions happen best
at a pH near the pK of His, i.e. ~ pH 7.

Question 24

What is the pH range at which chymotrypsin has little activity?

Answer 24

Below pH 7 and above pH 9.

Question 25

What are the two factors that can reflect an enzyme’s pH optima?

Answer 25

1) Active site acid-base chemistry
2) Sensitivity of overall protein conformation to charge distribution

Question 26

What happens to the conformation of chymotrypsin
above pH 9?

Answer 26

The conformation of chymotrypsin is disrupted because structurally important amino groups become unprotonated and uncharged, resulting in little activity.

Question 27

What is a multifunctional enzyme?

Answer 27

combination of different enzymes into a single polypeptide

Question 28

Enzymes in a common _________ are often physically associated with one another, either by __________________ interactions, or by binding to a common __________________.

Answer 28

pathway
direct binding
“scaffold” protein

Question 29

What are allosteric effects?

Answer 29

Binding of a ligand at one site on a protein can lead to conformational changes that affect the binding of another ligand molecule at a different site.

Question 30

How does Ca++ binding to calmodulin affect its function?

Answer 30

It changes its conformation, allowing it to bind to target peptides on other proteins, thus regulating their structure and activity.

Question 31

What are G-proteins?

Answer 31

They exist in “on” (GTP bound) and “off” (GDP bound) conformations that interact differently with other proteins.

Question 32

What facilitates the switching of G-proteins from “on”
to “off”?
What facilitates the switching of G-proteins from “off”
to “on”?

Answer 32

GAPs
GEFs

Question 33

What is a GEF?

Answer 33

A GEF is a guanine nucleotide exchange factor

Question 34

What is a GAP?

Answer 34

A GAP is a GTPase activating protein

Question 35

What is phosphorylation?

Answer 35

Phosphorylation is a rapidly reversible covalent modification of protein structure.

Question 36

What is a kinase?

Answer 36

an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule.

Question 37

What is the target of a kinase or a phosphatase?

Answer 37

Frequently the target of a kinase or a phosphatase is another kinase or phosphatase, creating a “cascade” effect.