Enzymes do not obey Michaelis-Menten kinetics. What do they follow?
Sigmoidal activity curves
Michaelis Menten assumes one independent binding site and no interaction between sites, but allosteric enzymes usually have multiple subunits and interacting binding sites. because binding at one site affects the others, kinetics are different
sigmoidal due to cooperativity (positive homoallostery), at least two states: high and low activities (R and T states). Often associated with feedback inhibition\
coopertivity means binding of one substrate increases the affinity of other binding sites
i.e. first substrate binds, enzyme shape changes, other sites bind substrate more easily
homoallostery means the substrate itself acts as the regulator
T state = tense = low activity/affinity
R state = relaxed = high activity/affinity
What are effectors/modulators?
bind non-covalently to regulatory sites
homotropic/homoallostery: substrate act as effectors, may bind to active sites or regulatory sites
heterotropic/heteroallostery: non-substrate effectors, bind to regulatory sites
What are other features of allosteric enzymes?
many have quaternary structure, where each oligomer can exist in R or T state (high and low activity, respectively).
overall protein symmetry is maintains (all subunits are in the same conformation)
effectors may bind to regulatory subunit or regulatory sites on catalytic subunits
effectors may increase activity (positive) or decrease (negative
What is the symmetry model of allostery
ligands will bond to both states with different affinities, and binding shifts eq between T and R states, symmetry maintained
What is the sequential model of allostery
overall protein symmetry is not necessarily maintained (i.e. all subunits aren’t necessarily in the same conformation)
Describe Aspartate Transcarbamoylase (ATCase)
EC2.1.3.2
Quaternary structure
6 catalytic subunits
6 regulatory subunits
D3 symmetry
first step in CTP biosynth (activated by ATP, feedback inhibited by CTP (allosteric modulators)
generally follows the symmetry model, R and T structural states
carbonyl phosphate and aspartate -> (ATCase) -> N-carbamoyl aspartate -> (6 enzymatic reaction steps) -> cytidine triphosphate (CTP) (inhibits ATCase)
T state is favoured by CTP binding, R state is favoured by substrate binding
In Aspartate Transcarbamoylase (ATCase), binding of PALA stabilizes the R state in ATCase. Why?
Because PALA mimics substrate binding → shifts equilibrium toward active conformation
Competitive inhibitors
In Aspartate Transcarbamoylase, CTP is a what? ATP is a what? Aspartate is a what?
CTP → heterotropic inhibitor
ATP → heterotropic activator
Aspartate → homotropic activator
Describe phosphofructokinase
kinase (phosphotransferase, 2.7.1.11)
homotetramer (D2 symmetry)
each subunit contains an active site and a regulatory site
What does the relative activity vs concentration curve look like between fructose 6 phosphate and ATP, given AMP vs no AMP
fructose 6 phosphate: binds to active site, homotropic activator, sigmoidal relationship. no AMP lower, but same start and end points
ATP: two binding sites (active site and regulatory site), homotropic inhibitor. same start curve, same peak, no AMP looks like bell curve, AMP has a plateau
AMP: activator (heterotropic), same regulatory site at ATP
phosphoenolpyruvate: heterotopic inhibitor
Describe reversible covalent modification of polypeptides
covalent bond is formed between enzyme and chemical group, but the modification can later be removed by another enzyme, so its reversible
modifications often cause conformation (tertiary structure) changes, which activate enzyme, inhibit enzyme, change binding properties
1) phosphorylation (adding a phosphate group). commonly phosphorylated amino acids: Ser, Thr, Tyr, His
Enzyme+ATP→Enzyme-P+ADP
kinase adds phosphate, phosphatase removes phosphate
phosphate groups carry negative charge, create new electrostatic interactions, change protein shape. target seq must match the kinase active site and be available on PROTEIN SURFACE. multiple targets/protein kinases allow for finer control
2) adenylation (add AMP group). instead of just transferring phosphate, enzyme AMP attached. common site: tyrosine residues
Enzyme+ATP→Enzyme-AMP+PPi
3) ADP ribosylation: larger group, ADP-ribose, is attached to a protein. often used in cell signaling, bacterial toxins
4) palmitoylation. fatty acid, palmitate, attached to protein. purpose: anchors proteins to membranes
Protein kinase A recognizes the target sequence:
-x-R-[R/K]-x-[S/T]-B-
Which of the following sequences match the target sequence?
(x indicates any amino acid, B indicates hydrophobic)
A. -SRKKSVSS-
B. -NTRRSSILLY-
C. -QERRESF-
D. -TAGVRKASL-
E. All of these sequences match the target sequence.
E
Describe regulation of glycogen phosphorylase by phosphorylation
glycogen phosphorylase is regulated by reversible phosphorylation to control glycogen breakdown.
Glycogen phosphorylase catalyzes:
Glycogen→Glucose-1-phosphate
So it breaks down glycogen.
Because this controls energy release, the enzyme is tightly regulated.
2 forms of glycogen phosphorylase (enzyme exists in two interconvertible forms): phosphorylase b (less active, dephosphorylated) + phosphorylase a (more active, phosphorylated)
Phosphorylation activates glycogen phosphorylase.
activation pathway:
1) hormonal signals (epinephrine, glucagon) activate protein kinase A (PKA)
PKA phosphorylated phosphorylase kinase
2) phosphorylation converts phosphorylase kinase b to phosphorylase kinase a
3) active phosphorylase kinase phosphorylates
glycogen phosphorylase b + ATP
producing
glycogen phosphorylase a + ADP
enzyme is active and glycogen breakdown increases
4) phosphatase (phosphoprotein phosphatase-1 (PP1) removes phosphate
glycogen phosphorylase a -> glycogen phosphorylase b
inactivating the enzyme
Multiple phosphorylation sites in glycogen synthase allows for multiple pathways of regulation. Describe
Individual phosphorylation sites can be affected by different kinases
each phosphorylation site is associated with a reduction in activity (contrast to glycogen phosphorylase)
multiple phosphorylation will act in concert to reduce activity of glycogen synthase
Describe irreversible covalent modification
covalent change in polypeptide primary structure
zymogens (unaltered enzymes) are activated by modification by other enzymes
(such as digestive proteinases are modified by other proteinases)
enzymes must then be destroyed or inactivated
Describe how digestive proteases are activated from inactive precursors (zymogens) in the small intestine (to prevent the enzymes from digesting the pancreas itself)
1) trypsinogen (from pancreas) cleaved to endopeptidase (enterokinase) to trypsin (active)
2) trypsin activates more trypsinogen (positive feedback)
3) trypsin activates other digestive enzymes: such as chymotrypsinogen to π-Chymotrypsin (partially active) (trypsin cutes the peptide chain after residue 15, allowing the active site the form properly, including the oxyanion hole, stabilize the negatively charged oxygen in the tetrahedral intermediate)
4) π-Chymotrypsin undergoes additional cleavage steps to produce the fully active enzyme
π-Chymotrypsin to α-Chymotrypsin (three peptide chains held together by disulfide bonds)
Describe the structure of chymotrypsin
initial polypeptide is 245 amino acids long
cleaved into 3 chains during activation
disulfide bonds link the three chains to each other (one covalent structure)
10 cysteines form 5 disulfide bonds
What are the three important catalytic residues?
Ser195 – nucleophile
His57 – general acid/base
Asp102 – stabilizes His57
far apart in the primary sequence, but close together in the folded protein.
His activates Ser
Ser attacks the peptide bond
Asp stabilizes His
This arrangement allows the enzyme to perform covalent catalysis and acid–base catalysis simultaneously.
