Lecture 23 - Protein function and regulation Flashcards
Common activity of all proteins
Binding
Name of what proteins bind to and consequence of binding
Bind to ligand -> Conformational change that may result = the protein does its function
2 important properties in ligand-binding
Specificity and Affinity
What do we mean by the specificity of a protein
Its abiliy to bind a particular ligand even when in presence of many irrelevant molecules
What do we mean by the affinity of a protein and how is measured
Strength of binding. Dissociation constand (Kd). Lower Kd = stronger binding
What makes binding possible (how is it possible if bonds between molecules are very weak)
Summation of all interactions between 2 protein surfaces makes binding
4 things that are good for binding of 2 surfaces
1) Surfaces have complementary shapes
2) H bonds
3) Complementary charges (plus one side, minus other)
4) Hydrophobic interactions
Best molecules for specificity and affinity + how they bind
Antibodies. Bind with CDR (complementarity determining region)
Where CDR found on antibody/what it’s made of
On both ends of the Y, made of loops of heavy chain and light chain that are highly variable
Enzymes : Special thing about their ligands
Are the substrates of the reactions they catalyze
General scheme of reaction from energy POV
Reactants energy, Transition state (activation energy). Products energy
What enzymes change in a reaction (2)
1) Reduce activation energy
2) Therefore, increase reaction rate
where on enzyme do substrate (ligand) binding and reaction catalysis occur 2 components of this region
In enzyme’s active site. Has a substrate binding site and a catalytic site
How rigid is the binding of an enzyme to its substrates + name of phenomenon by which they fit together
Fluid. There is some molecular flexibility which allows an induced fit of substrate in its binding site
What makes up an enzyme’s catalytic site and substrate binding site
Amino acids that are close by on its surface (3D shape) but that could be far in the linear polypeptide
What are proteases
Proteases cleave proteins (hydrolyze peptide bonds).
what kind of protease trypsin is + why
Trypsin = serine protease cause catal. mechanism involves key serine (serine 195) w/ OH group
Specific activity of trypsin (where it cuts)
Cuts between lysine and arginine residues
Special name of substrate binding site in trypsin
Side-chain-specificity binding pocket
What goes in the side-chain-specificity binding pocket and how it’s held there
Arg or Lys positively charged side chain. Held by negatively charged aspartate
Beside interaction on the substrate binding site, how can interactions also occur between substrate and enzyme
Possible interactions between enzyme and substrate backbones (H bonds) elsewhere
What determines specificity of the enzyme
ONLY the interaction between peptide and substrate binding site (aspartate and side chain of residue at Arg position)
2 other serine proteases like trypsin but with different specificities
Chymotrypsin and elastase
Chymotrypsin substrate binding site (side-chain-specificity binding pocket)
Contains a serine so less specificity than trypsin for basic residues
Elastase substrate binding site (side-chain-specificity binding pocket)
Contains bulky amino acids like valine so cleaves beside a.a s with small side chains
3 key amino acids in the catalytic mechanism of trypsin and other serine proteases
Ser 195, his 57 and asp 102
First step of serine proteases catalytic mechanism
Cleavage of peptide bond to free P2 N-termini and formation of an acyl complex
Second step of serine proteases catalytic mechanism
Hydrolysis of acyl enzyme complex
Intermediate in first and second step of serine proteases catalytic mechanism + special name and why
Tetrahedral intermediate called an oxyanion cause it has a single bond to an oxygen that is negatively charged
First general rule of enzyme catalysis
Enzyme catalysis based on stabilized binding of transition state
Second general rule of enzyme catalysis
Enzyme catalysis based on 3D organization of key amino acids in the active site
First common observation/pt of interest about enzymes
May break down a reaction into several sub-reactions
Second common observation/pt of interest about enzymes
Enzyme’s active site chemistry may be pH dependent
Why pH influences enzyme activity (so why they have an optimal pH)
1) pH influences active site acid-base chemistry
2) pH influences conformation of whole protein
Exemple of enzymes that adapted to work at low pH and how they did it
lysosomal hydrolases -> use different catalytic mechanism
Why chymotrypsin doesn’t work at pH lower than 8
His-57 becomes already protonated so can’t steal proton (1st step in mech.)
What Michealis-Menten enzyme kinetics show
Rate of an enzymatic reaction can be expressed as function of substrate concentration
What is the Vmax
Maximal rate of catalysis given saturating amounts of substrate
What influences Vmax
Amount of enzyme and turnover number
What is the turnover number
maximum number of substrate molecules converted to product per enzyme molecule per second.
V max formula
Enzyme (site) concentration * turnover number
What is Km
substrate concentration that supports a rate of catalysis equal to half the Vmax
Does the enzyme qt influence the Km
No. No matter the Vmax (influenced by enzyme qt) Km is always the same for a given enzyme-substrate pair
Why Km is constant for an enzyme-substrate pair
Binding affinity independent of concentrations and depends only on chemical properties of enzyme + substrate
Ways to accelerate effect of enzymes involved in a common pathway (of reactions) (3)
Binding of enzymes together
Binding of enzymes on a common scaffold
Link enzymes’ genes to make a common multienzyme complex
Name of binding of many enzymes together and utility
Multienzyme complexes. Products from a catalyzed reaction are easily available for next one
Name of enzymes’ genes linked into one enzyme and characteristic
Multifunctional enzyme. Many different domains and catalytic sites with related reactions
What are allosteric effects
Change of protein conformation at a site other than ligand binding site
Consequence of allosteric effects
Protein may be able to accomplish new function
2 major manifestations
1) Cooperative binding of substrates to multimeric protein complexes
2) Conformational switches in regulatory proteins in response to post-transl. modif (or ligand binding obviously)
Example of cooperative binding in the blood
When one O2 molecule binds hemoglobin, increases Hb affinity to other O2 molecules and they will bind more readily (conformational change)
Shape of O2 binding curve and what each axis represents
Sigmoidal shape (S-shape) on graph pO2 on X and Saturation of Hb w/ O2 on Y axis
2 examples of allosteric switches
Noncovalent binding of Ca 2+ and GTP
Example of allosteric switch w/ calcium binding
When calmodulin binds calcium, it can adopt proper shape to bind target peptides (thus regulating their structure/activity)
Explanation of allosteric switch w/ GTP
Proteins that are GTPases regulated by GTP and can hydrolize it
GTP -> GDP : becomes inactive and step accelerated by GAP (GTPase activating protein)
GDP -> GTP : becomes active and step accelerated by GEF (guanine nucleotide exchange factor)
On what structure of a protein phosphorylation/dephosph. of a protein is done and bond nature
Phosphorylation of amino acid SIDE CHAINS -> COVALENT bond
How phosphorylation/dephosph. can affect protein activity
Phosphorylating protein can activate it and dephosphorylating it can inactivate it
What proteins regulate phosphorylation /dephosphorylation and how
Protein kinases phosphorylate (add a Pi to protein and an ATP becomes ADP), protein phosphatases dephosphorylate (remove Pi w/ water)
How many protein kinases we have
More than 500 diff. kinases in human genome
