Sept 13 Protein Function and Regulation Flashcards
what activity is the basis of the many diverse functions of proteins?
binding
proteins bind to one another, macromolecules, small molecules and ions
what is the molecule to which a protein binds?
the ligand
what happens in some cases when the protein binds to its ligand?
ligand binding can change the conformation of the protein
ligand induced conformational change is integral to the mechanism of action of many proteins
what are the two most important properties in ligand binding?
specificity and affinity
what is specificity?
the ability of a protein to bind to only one particular ligand, even in the presence of a big excess of irrelevant molecules
what is affinity?
the tightness/strength of binding, expressed as a dissociation constant Kd
stronger interaction –> lower Kd
what does protein binding specificity arise from?
arises from numerous interactions which are individually weak but collectively strong
binding is an interaction between complementary molecular surfaces
how do antibodies bind to their antigens and what allows them to do so?
antibodies bind to their antigens with high specificity and high affinity
antibodies two identical heavy chains and two identical light chains linked by disulfide bonds
antigen binding surface or CDR (complementarity determining region) involves multiple protein loops from both the heavy and light chains
what are enzymes and what is their ligand called?
extremely diverse class of catalylitcally active proteins whose ligands are the substrates of the reactions they catalyse
where does substrate binding and reaction catalysis occur?
occurs at the enzymes active’s site
the active site includes the substrate binding site (complementary to the specific substrate)
and the catalytic site which carries out the chemical reaction once the substrate is bound
what is Vmax?
the maximal rate of catalysis given saturating amounts of substrate
depends on the amount of enzyme and how fast it can work
turnover number–> number of enzymatic cycles per second at top speedw
what is Km?
the substrate concentration that supports a rate of catalysis equal to one half of the V max
depends on, and is a measure of the affinity of enzyme:substrate binding
how do the values of Vmax and Km differ when you the concentration of enzymes changes?
the Vmax is higher, proportional to how many more enzymes were added
however the Km stays the same, because in general, binding affinity is independent of concentrations, but depends only on the chemical properties of the enzyme and the substrate?
what does the fact that enzymes exhibit pH optima reflect?
- evidence of active site acid base chemistry
- sensitivity of the overall protein to charge distribution
what happens to enzymes in a common pathway? (what is metabolic coupling)
there are three ways
1. the enzymes can become associated together into a quaternary structure which is much more efficient
each enzyme is coded for by a different gene
2. the enzymes can bind to a common “scaffold” protein
3. by evolution, enzymes can combine into a single polypeptide, coded for by a single gene
a multifunctional enzyme which has different domains(!!!)
what is allosteric effect?
binding of a ligand at one site of a protein can lead to the conformational change that affect the binding of another ligand at a different site
can be used to control protein activity, turn it “on” or “off”
in what other ways can protein conformation and activity be modified?
through phosphorylation and dephosphorylation
phosphorylation of amino acid side chains is a rapidly reversible covalent modification of protein structure –> post translational modification
what needs to happen for high affinity and highly specific interactions to take place?
the shape and chemical properties of the binding site must be complementary to those of the molecule
what is the induced fit model?
substrate binding site is not rigid, but flexible
and that substrate binding induces the enzyme to change shape and consequently bind more strongly to the transition state
what is the enzyme substrate complex?
the bound substrate to the enzyme
the ES complex is in equilibrium with the unbound enzyme and substrate and is an intermediate step, followed by the conversion of the substrate to the product
how do enzymes catalyse the process of a reaction?
they divide the it into multiple, discrete chemical reactions, in which the product of one reaction is the substrate for another
different ES complexes are generated before the final product
what is a cofactor/prosthetic group?
helpergroup, non polypeptide small molecule or ion that is bound in the active site and plays an essential role in the reaction mechanism
also called coenzymes
some (not all) are chemically modified during the reaction and therefore need to be replaced or regenerated
what are enzyme inhibitors?
bind to active sites and disrupt catalytic reactions
what are competitive inhibitors?
bind directly on an enzyme’s binding site and this compete directly with the normal substrate
what are non competitive inhibitors?
interfere with enzyme activity, in other ways, binding somewhere that is not the substrate binding site and change the enzyme’s conformation
what does negative allostery often involve? and what does it do?
involves the end product of a multistep biochemical pathway
that end product will bind to the allosteric site of a enzyme that catalyses an early, rate controlling step in that pathway, therefore reducing its activity
excessive buildup of the product is prevented
it is called end-product inhibition
what is cooperativity and its effects?
the influence (+ or -) that the binding of a ligand at one site has on the binding of a molecule of the same type of ligand on a differ site
what is an example of cooperativity?
hemoglobin is a classic example of positive cooperativity
the binding of a single ligand (oxygen) increases the affinity of hemoglobin for the next oxygen molecule
(binding of one ligand increases sensitivity for another ligand)
cooperativity permits hemoglobin to take up oxygen very efficiently in the lungs and unload in tissues
provides a selective evolutionary advantage
what is phosphorylation?
reversible addition of a phosphate group from ATP to a hydroxyl group of a side chain of a serine threonine or a tyrosine residue
changes the protein activity
what enzymes are involved in phosphorylation and dephosphorylation?
phosphorylation is catalysed by kinases
dephosphorylation is catalysed by phosphatases
what can phosphorylation and dephosphorylation influence? (5)
- location of a protein within cells
- structure and intrinsic activity
- its ability to bind to other molecules
- its ability to undergo further covalent modifications
- its stability
which proteins are regulated by kinase and phosphatase modifications?
all classes of proteins
how is a cascade effect created by phosphorylation and dephosphorylation?
the target of a kinase or phosphatase is yet another kinase or phosphatase
what is the definition of binding?
an interaction between complementary molecular surfaces
explain the mechanism of serine protease trypsin as an example of enzyme-substrate binding and catalytic site specificity:
proteases hydrolyse peptide bonds in polypeptides
the serine proteases are a family of proteases whose catalytic mechanism involved a serine residue in the catalytic site
trypsin hydrolyses peptide bonds (digestion) adjacent to arginine and lysine, which are large basic side chains
proper substrate binding only occurs when the substrate amino acid side chain “fits” into the negatively charged pocket within the substrate binding side
the different substrate recognition pockets in enzymes define their specificity
positive arginine interacts with the negative side chain from the enzyme
arginine stays in position long enough for catalysis to happen
why do proteins fold?
to bring distant amino acids into proximity, in a way that is chemically effective
explain the mechanism of trypsin in detail (see slide 9 of lecture)
Step 1: cleavage of a peptide bond with formation of a covalent substrate enzyme complex
a. oxygen from Serine side chain attacks the carbonyl carbon on the polypeptide backbone
this is facilitated by the fact that histadine is basic, it can readily accept protons and obtain a positive charge
b. the oxygen and carbon are now covalently bonded, the carbon is now part of the enzyme (transition state)
electrons that form the peptide bond pull back hydrogen proton from histadine
c. by condensation, fragment can now break away, while part with N terminal is still attached
step 2: hydrolysis of acyl enzyme complex
d. with water, the bond between the oxygen (from the enzyme) and the carbonyl carbon is hydrolysed
the electrons on the oxygen from water attack the carbonyl carbon
again, this is facilitated by histadine
e. a proton from water is picked up by histadine
f. the bond between the oxygen and the carbonyl carbon is broken, and the protein diffuses away
the active site is regenerated like at the beginning
what are two allosteric switches that are widely used to control protein activity?
noncovalent binding of Ca2+ and GTP
what is the effect of Ca2+ binding to calmodulin?
it changes its conformation, allowing it to bind to target peptides on other proteins, thus regulating their structure and activity
calmodulin can turn on other proteins
Ca2+ activates calmodulin, which activates other proteins
what are the two settings of G-proteins and what is their effect?
“on” (GTP bound) and “off” (GDP bound) conformations that interact differently with other proteins
switching from on to off is facilitated by GAPs and switching from off to on is facilitated by GEFs