protein function and regulation

ligand

molecule to which a protein binds

specificity

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

affinity

the tightness, or strength, of binding, expressed as dissociation constant (K_d)

relationship between interaction strength and dissociation constant

stronger interaction → lower K_d

protein binding

results from an interaction between complementary molecular surfaces

structure of antibodies

• two identical heavy chains and two identical light chains

• individual polypeptides (chains) are held together through disulfide bonds

antigens

molecules recognized by antibodies

CDR (complementarity determining region)

also known as antigen-binding surface, region on antibody which is complimentary to the antigen and involves multiple protein loops from both the heavy and light chains

enzymes

• proteins or RNA

• catalyze making or breaking substrate covalent bonds

• ligands include the substrates of the reactions they catalyze

• accelerate the rate of reaction by reducing the free energy of the transition state

active site

contains substrate binding site and active site, where the substrate binds to the enzyme and the reaction catalysis occurs

V_max

• the maximal rate of catalysis given saturating amounts of substrate

• depends on the amount of enzyme and how fast it can work

  • turnover number, enzymatic cycles per second at top speed

K_m

• the substrate concentration that supports a rate of catalysis equal to ½ of the V_max

• provides a measure of the enzyme:substrate binding affinity

assembly of multienzyme machines

1. for enzymes free in solution, reaction intermediates diffuse from one enzyme to the next, which may be inherently slow

2. multisubunit enzyme complex formed by a scaffold protein minimizes or eliminates the substrate diffusion time

3. some enzymes are fused at the genetic level, becoming domains in a single polypeptide chain

proteases

hydrolyze peptide bonds in polypeptides

serine proteases

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

trypsin

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

• proper substrate binding only occurs when the substrate amino acid side chain fits into a negatively charged pocket within the substrate binding site

protease binding pocket

differences in the substrate recognition pocket in the related enzymes define their differing specificities

elastase binding pocket

obstructed by bulky valine side chains, so elastase cleaves adjacent to amino acids with small side chains (glycine, alanine)

catalytic site of trypsin

uses a 2 step catalytic mechanism involving 3 amino acid side chains: Asp-102, His-57, and Ser-195

peptide cleavage catalyzed by trypsin

1. cleavage of peptide bond with formation of a covalent substrate-enzyme complex (Ser-195 acyl enzyme)

2. hydrolysis of acyl enzyme complex

both subreactions depend on His-57’s ability to bind and to release a proton

acid-base catalysis

pH dependent, requires a particular ionization state (protonated or nonprotonated) of one or more amino acid side chains in the catalytic site

pancreatic serine proteases (chymotrypsin)

activity is optimum at pH=8, pH<9 required for proper protein conformation

lysosomal hydrolases

pH of ~4.5 is optimum, matches the low internal pH in the lysosomes in which they function

allosteric effect

results from the binding of a ligand at one site on a protein leading to conformational changes that affect the binding of another ligand at a different site

allosteric regulation

conformational switches in regulatory proteins in response to ligand binding or post-translational modification

calmodulin

example of allosteric regulation:

Ca²⁺ binding changes its conformation, allowing it to bind to target peptides on other proteins

G-proteins

example of allosteric regulation:

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

• switching from “on” to “off” is facilitated by GAPs (GTPase activating proteins)

• switching from “off” to “on” is facilitated by GEFs (guanine nucleotide exchange factors)

post-translational modifications

• changes in the chemistry of proteins that occur after synthesis

• modifications of protein conformation and activity can occur through phosphorylation and dephosphorylation

protein kinases

phosphorylate amino acid side chains

protein phosphatases

catalyze the removal of a phosphate group (dephosphorylation) of amino acid side chains