Enzyme Regulation Flashcards
What is a double reciprocal plot? What are the axes and intercepts?
Lineweaver-Burke Plot - used to determine mechanism of an inhibitor’s effect on rxn (type of inhibition)
Axes: inverse initial velocity (y) vs inverse substrate concentration (x)
Intercepts: X-intercept = 1/Vmax, Y-intercept = -1/Km
How do the V/[S] plots differ when a competitive, uncompetitive, or noncompetitive inhibitor is added?
Competitive: slope changes, y-intercept same
Uncompetitive: slope same, y-intercept changes
Non-competitive: slope changes, y-intercept changes
Why do Vmax and Km change/don’t change with each inhibition type?
Competitive: Km incr b/c incr substrate needed to outcompete inhibitor since more binding sites taken by inhibitor; Vmax doesn’t change b/c inhibitor outcompeted to point that doesn’t affect enzyme
Uncompetitive: Km decr b/c substrate tightly bound (high affinity), off-rate is lower as affinity incr; Vmax decr b/c product formation decr
Non-competitive: Km doesn’t change b/c substrate and inhibitor bind at different sites; Vmax decr b/c product formation decr
What makes an inhibitor irreversible? What has to happen in such situations?
Binding of inhibitor to enzyme is permanent
FINISH
Suicide substrate inhibitor
Irreversible inhibitors that begin initial steps of catalysis but form a covalent intermediate that kills enzyme
Ex: nerve gas, roach spray
How are enzymes regulated in a cell?
Allosteric regulation - enzyme/regulatory protein binds ligand at site different from active site
Kinetic control - availability of substrate
Covalent modifications - faster regulation (WHY FASTER)
HOMOTROPIC VS HETEROTROPIC REGULATION
What mechanisms affect an enzyme’s activity but not abundance?
What mechanisms affect where an enzyme and substrate co-occur in a cell?
What mechanisms are strictly kinetic effects?
What kinds of covalent modifications can occur? Exs?
Phosphorylation
Adenylylation
Acetylation
Myristoylation - attaching hydrophobic fatty acid to enzyme to change localization in a cell
Ubiquitination
ADP-ribosylation
Methylation
EXAMPLES
Why do regulatory enzyme kinetics differ from non-regulatory enzymes?
Differences btw regulatory enzyme kinetics and non-regulatory enzymes
Why are the velocity curves of regulatory enzymes sigmoidal?
What types of regulation are observed for ATCase?
Aspartate transcarbamoylase (ATCase - Important regulatory enzyme in pyrimidine biosynthesis)
Homotropic regulation - Increases in carbamoyl aspartate and phosphate cause gradual change from inactive (T state) to active (R state)
Positive heterotropic modulation - ATP stimulates ATCase by building up sufficient CTP (cytosine - pyrimidine), energy costing pathway
Negative heterotropic modulation - CTP inhibits ATCase b/c excess pyrimidines not being used, signals need to shut down synthesis; regulatory subunits likely to bind CTP
What types of regulation are observed for glycogen phosphorylase?
Glycogen phosphorylase - moves glycogen (stored glucose) when urgently needs it for glucose oxidation/energy production (low nutrition, prolonged exercise)
Allosteric regulation - AMP levels rise as cell struggling to generate enough ATP to continue functioning (low energy), activates glyc phosphorylase
Covalent modification - phosphorylation of serine-14 shifts equilibrium to more active R state (phosphorylase A), phosphorylated by phosphorylase kinase activated by hormone signaling
Hormonal regulation - epinephrine/adrenaline (moves muscle glycogen for sudden need for muscle contraction and ATP) and/or glucagon (moves glycogen b/c of sudden low blood sugar) activates adenylyl cyclase;
kinase cascade (Incr cAMP activates protein kinase A -> PKA activates phosphorylase kinase -> phosphorylase kinase phosphorylates glycogen phosphorylase -> glucose released from glycogen to generate needed energy)
