Enzymes Flashcards
Transition state analogs
Usually man-made…simulate a real transition state and bind to a specific enxyme to prevent products from being made
Usually competitive inhibitors
Ex: HIV protease inhibitors and tamiflu
Oxidoreductases
Catalysts in oxidation/reduction reactions
Glycolysis and krebs cycle
Transferase
Transfer one functional group from one molecule to another molecule
Hydrolases
Use water to cleave one molecule into two molecules
Lyases
Work by adding or removing water, ammonia, or CO2 (but do not make 2 molecules from one)
Isomerase
Intramolecular rearrangements
Ligases
Catalyze high energy reaction using ATP…to join two molecules together
DNA ligase
Initial velocity will increase linearly as…
Amount of enzyme is increased
Initial velocity vs. [substrate]
Yields a bowed graph
Vmax = max velocity
Km = [subrate] at 1/2 of Vmax
Lower the Km the ? The affinity of the substrate to bind to the enzyme…
Higher
E + S (k1–>)(K2)E+P
Km =?
(K1+K2)/K3
Michaelis - menten equation
Velocity = Vo = (Vmax*[S])/(Km+[S])
At low [substrate]
What can be changed in MM equation
Adding [S] in denominator
At high concentration Vo = ?
Vmax
When [S]=Km
Then…
Vo=Vmax/2
Kcat =
Constant that describes the limiting rate of any enzyme at substrate saturation
Kcat = Vmax/[total enzyme]
Value in sec(-1) units
Or…the amount of substrate that is turned into product for one enzyme molecule when it is saturated
Regulation via non-covalent changes
Fastest form of regulation
Inhibit activity by REVERSIBLE binding to enzymes
Can either increase or decrease activity of an enzyme that is already there
Regulation via covalent changes
Proteolytic processing, covalent modifications, irreversible inhibitors
Ex: phosphorylation/de-
Proteolytic processing = irreversible
What is the MM plot NOT useful for?
Determining the type of inhibition
Lineweaver-Burk PLot
Used to determine type of reversible inhibition is occuring
LB plot
X-intercept
Y-intercept
Slope
-1/Km
1/Vmax
Km/Vmax
Competitive inhibition
Binds at the active site
Can be reversed by….increasing [substrate]
Km increase as [I] increases = means to more inhibitor is added…the more substrate is needed to keep level of activity
Vmax = stays same!!! —> if enough substrate is added … it will overwhelm the effect of the inhibitor
**enzyme usually has a higher affinity for the inhibitor
Drugs: statins, methotrexate
Mixed inhibition
Noncompetitive inhibition
Mixed = binds to a site distant from the active site to both E and ES
Km = increases Vmax = decreases
Most ‘feedback inhibitors’ are these
Noncompetitve = special kind of mixed
Km = no change Vmax = decreases
Bind to site distinct from active site and binds to either E or ES
Ex: organophosphate pesticides
Uncompetitive inhibitors
Site distant from the active site
ONLY when the enzyme is in the ES complex
Both Km and Vmax are lowered
Allosteric regulation
Important in metabolic pathways
Changes in one region/subunit of protein alter the conformation of other regions of subunits
Both activation and inhibition
Final products of pathway are often allosteric inhibitors of a key reaction in the pathway
S-shaped plot!!!
Always have multiple subunits
More likely to effect Km than Vmax
Post-transitional modifications to proteins
Covalent modifications
Most imporant = phos, acetylation, ubiq
Cholera toxin
ADP ribosylation
Permanently ADP-ribosylates an Arg. In the G-alpha subunit of the G-protein in the intestinal epithelial cells
—> results in cAMP production
—> secretion of water, Na+, K+, Cl-, HCO3- into the lumen —> dehydration
Caffeine = same effect but is a reversible inhibitor
O-linked N-acetylglucosamine
Reversibly inhibits PK1 in response to hypoxia or other cellular stress in cancer cells (not in non-cancerous)
This will shuttle more glucose to the PPP
Where it can make DNA precursors and NADPH to protect cancer cells from oxidative stress…
Prolyl and asparagunyl hydroxylation
Important in oxygen sensing by the transcription factor HIF
Tells the cell its becoming ischemic
Proteolytic processing and zymogen activation
Breaking of bonds —> irreversible regulation
Common in digestion
Many proteins are made in an inactive form…zymogen
Then can be turned into active proteases once they arrive at desired destination via proteolytic cleavage
Irreversible inhibitors
Different than transition staet analogs
= form covalent bond with substrate during catalyitc process which kills/inactiveates the enzymes
Alpha-1-antitrypsin deficiency (A1AD)
May develop emphysema during their thirties even without smoking
Impair live function as well - most common cause for liver transplant in infants
Type of Serpin
Antithrombin III
Also a serpin
Defects lead to increased formation of clots in veins and arteries
