Lecture 7 Information Flashcards
How do enzymes with more than 1 substrate bind them?
Can form a tertiary complex to bind both at the same time
Can interact with multiple substrates in an ordered manner (interact with one substrate before the other)
Can interact with multiple substrates in a ping pong manner (one is converted to product then the other is converted to product)
Binding affinity for more than 1 substrate
Enzymes will have a different binding affinity (Km) for each substrate they bind
2 main classes of enzyme inhibition
Reversible interaction
Irreversible interaction
Competitive inhibition
a type of reversible inhibition
inhibitor binds at the active site and forms EI complex
Kinetic effects of competitive inhibition
Vmax stays the same because you can flood the system with a high amount of substrate
Apparent Km increases
Uncompetitive inhibition
a type of reversible inhibition
inhibitor binds to the ES complex apart from the active site
Kinetic effects of uncompetitive inhibition
Vmax decreases
Apparent Km decreases
Mixed inhibition
a type of reversible inhibition
inhibitor binds to free enzyme or ES complex
Noncompetitive inhibition
a special type of mixed inhibition
the affinity of the inhibitor to bind to the free enzyme or to the ES complex is equal
Kinetic effects of noncompetitive inhibition
Vmax decreases
Apparent Km stays the same
Irreversible inhibition
bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity
Example of irreversible inhibition
penicillin binds irreversibly to transpeptidase to prevent bacterial cell wall from being generated
Transition-state analogs
A type of irreversible inhibition that uses weak interactions
Creates a better transition state with more weak interactions that prevents the substrate from binding
Suicide inactivators
combines irreversibly to the enzyme through covalent interactions
hijack the normal enzyme reaction mechanism
Regulatory enzymes
exhibit increased or decreased catalytic activity in response to certain signals
they regulate and they also are regulated
Allosteric enzymes
function through reversible, non-covalent binding of regulatory components
Allosteric modulators/effectors
noncovalently bind to allosteric enzymes to either inhibitory or stimulatory regulate
Different ways to regulate enzymes
1) Allosteric enzymes
2) Covalent modification
3) Separate regulatory proteins
4) Proteolytic cleavage
Proteolytic cleavage
activates enzymes through removing peptide segments
this is irreversible unlike allosteric regulation
How do allosteric modulators work cooperatively?
conformational changes induced by one or more modulators interconvert more-active and less-active forms of the enzyme
Homotrophic enzyme
the modulator is the substrate and the active site is the regulatory site
Feedback inhibition
type of inhibition where the final product comes back to the first enzyme in a product and binds to it
binds to the first enzyme to save energy/materials
Heterotophic enzyme
the modulator is a molecule other than the substrate and the active site is not the regulatory site
Are enzymes turned completely off or on?
No! This is rare. Most are turned up or turned down in levels
Are regulatory sites specific?
yes, each regulatory site is specific for its modulator
How are allosteric modulators different from inhibitors?
Inhibitors do not necessarily mediate conformational changes between active and inactivate enzymes
Shape of allosteric enzymes on Michaelis-menten plot
Sigmoidal shape which reflects cooperative binding between multiple subunits
What do we call the Km of allosteric enzymes?
K(0.5)
have to do this since it doesn’t follow normal hyperbolic shape
represents substrate concentration giving half-maximal velocity
Covalent modifications of enzymes
examples: methylation, ribosylation, acetylation, adenylation, ubiquitin tag, phosphylation
Ubiquitin
entire protein that is covalently added to enzyme to tag it for destruction by a proteasome
Kinases
take a phosphate group (often from ATP) and use it to add phosphoryl groups to specific amino acids
What amino acids do kinases add to?
Ser, Thr, Tyr or sometimes His
How can phosphorylation effect an enzyme
can turn off an enzyme through the repulsion of negatively charged groups
can turn on an enzyme through the attraction of positively charged and H-bonds
Tyrosine kinases
important in cell signaling and associated with the cell membrane
often inactive, but when you bind a ligand the two subunits of the kinase come together
now, this kinase is activated and auto-phosphorylates more tyrosine kinases
now there are all these kinases that can turn on more enzymes
protein phosphatases
removes phosphoryl groups from same target proteins
When glycogen is phosphorylated what happens?
it releases a glucose-1 phosphate that can be used by the bloodstream
this transformation is catalyzed by glycogen phosphorylase
Is glycogen phosphorylase a kinase?
No
because it does not use ATP or any other nucleotide triphosphate to transfer phosphoryl group
Phosphorylase a
more active form of glycogen phosphorylase
Phosphorylase b
less active form of glycogen phosphorylase
Phosphorylase phosphatase
removes phosphoryl groups from phosphorylase a to make the enzyme less active in b form
Phosphorylase kinase
adds ATP to phosphorylase b to make the enzyme more active in a form
How is glycogen breakdown regulated?
by various ratios of phosphorylase a and phosphorylase b
Consensus sequences
common structual motifs where Ser Thr or Tyr residues are found to phosphorylate
Zymogen
inactive precursor that is cleaved to form an active enzyme
this is irreversible activation
proteases like chymotrypsin, trypsin, and pepsin have zymogen precursors to not destroy proteins within the cell
name does not apply to all precursors
What are most inactive precursors generally called?
proproteins or proenzymes
Proteolytic cleavage
changes zymogen to active chymotrypsin and trypsin enzymes
How do we regulate proteases once they are turned on?
Need another protein to interact with them
regulated through protein-protein interactions
Trypsin
cleaves after positive charges
breaks down proteins in the gut
Chymotrypsin
cleaves after aromatic rings
Glucagon
hormone that stimulates the process of glycogen breaking down into glucose 1-phosphate
Insulin
hormone that inactivates the process of glycogen breaking down into glucose 1-phosphate
decreases blood-sugar levels
Size of allosteric enzymes
typically bigger enzymes with more than one polypeptide chain
What are the zymogen’s called?
ex: trypsinogen is the inactive form of trypsin
Why are certain enzymes kept a low pH even when the cell is at a neutral pH? Like lysozomes?
So if the lysosome breaks open, the enzymes will have little activity in the neutral pH
ATCase
has homotrophic and heterotrophic regulation
ATCase has a sigmoidal curve cause has cooperative binding
Why does phosphatase not generate ATP when it removes phosphoryl?
requires too much energy
just cleaves a Pi
PKA
protein kinase a
sticks a phosphate onto inactive form of phosphorylase kinase to activate it
Hyperglycmic
too high blood-sugar levels
pancreas picks up on this signal and triggers insulin to drop glucose
Hypoglycmic
too low blood-sugar levels
pancreas picks up on this signal and triggers increase of glucose released (glucagon)
What does glucagon do?
inceases cAMP which activates PKA
PKA then activates phosphorylase kinase which can then change phosphorylase b to phosphorylase a
