Protein function and regulation Flashcards
What is the definition of a ligand?
The molecule to which a protein binds.
What is the importance of specificity in ligand-binding?
It allows a protein to bind only one particular ligand, even in the presence of a vast excess of irrelevant molecules.
What is affinity in ligand binding?
The tightness, or strength of binding, expressed as
dissociation constant (Kd).
What is the relationship between the strength of
interaction and Kd?
The stronger the interaction, the lower the Kd.
How does protein binding specificity arise?
It arises from numerous interactions which are individually weak, but, if numerous, collectively strong.
What is binding in molecular interactions?
It is an interaction between complementary molecular surfaces.
What are antibodies? (physical)
Antibodies contain two (identical) heavy chains and
two (identical) light chains.
What is the antigen-binding surface of antibodies?
The antigen-binding surface or CDR (complementarity determining region) involves multiple protein loops from both the heavy and the light chains.
What are enzymes?
Enzymes are an extremely diverse class of catalytically active proteins whose ligands include the substrates of the reactions they catalyze.
What is the active site of an enzyme?
Substrate binding, and reaction catalysis, occur at the enzyme’s active site.
Where does substrate specificity arise from?
Substrate specificity arises from the substrate binding site
What is Vmax?
Vmax is the maximal rate of catalysis given saturating amounts of substrate.
What is the turnover number?
The enzymatic cycles per second at top speed.
What is Km?
Km is the substrate concentration that supports a rate of catalysis equal to one-half of the Vmax.
What does Km depend on, is a measure of?
the affinity of enzyme:substrate binding
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.
4‐fold higher
same
independent
chemical
What do proteases do?
hydrolyze peptide bonds in polypeptides
What are the serine proteases?
The serine proteases are a family of proteases whose catalytic mechanism involves a serine residue in the catalytic site.
What is the mechanism of action of trypsin?
(hydrolyzes what? and what are the 2 steps?)
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
What amino acid side chains are involved in the trypsin mechanism?
catalytic mechanism involving 3 amino acid side chains Asp-102, His-57, and Ser-195.
What is the role of the negatively charged pocket in
the substrate binding site of trypsin?
Proper substrate binding only occurs when the substrate amino acid side chain “fits” into a negatively charged pocket within the substrate binding site.
Differences in the substrate recognition pocket in the related enzymes define their differing ___________.
specificities
What is the pH range at which trypsin subreactions reactions happen best?
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.
What is the pH range at which chymotrypsin has little activity?
Below pH 7 and above pH 9.
What are the two factors that can reflect an enzyme’s pH optima?
1) Active site acid-base chemistry
2) Sensitivity of overall protein conformation to charge distribution
What happens to the conformation of chymotrypsin
above pH 9?
The conformation of chymotrypsin is disrupted because structurally important amino groups become unprotonated and uncharged, resulting in little activity.
What is a multifunctional enzyme?
combination of different enzymes into a single polypeptide
Enzymes in a common _________ are often physically associated with one another, either by __________________ interactions, or by binding to a common __________________.
pathway
direct binding
“scaffold” protein
What are allosteric effects?
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.
How does Ca++ binding to calmodulin affect its function?
It changes its conformation, allowing it to bind to target peptides on other proteins, thus regulating their structure and activity.
What are G-proteins?
They exist in “on” (GTP bound) and “off” (GDP bound) conformations that interact differently with other proteins.
What facilitates the switching of G-proteins from “on”
to “off”?
What facilitates the switching of G-proteins from “off”
to “on”?
GAPs
GEFs
What is a GEF?
A GEF is a guanine nucleotide exchange factor
What is a GAP?
A GAP is a GTPase activating protein
What is phosphorylation?
Phosphorylation is a rapidly reversible covalent modification of protein structure.
What is a kinase?
an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule.
What is the target of a kinase or a phosphatase?
Frequently the target of a kinase or a phosphatase is another kinase or phosphatase, creating a “cascade” effect.
