Enzyme Kinetics 1 and 2 Flashcards
frequent drug target
enzymes, enzyme activity
why enzymes
biological reactions are too slow
(sucrose -> CO2 + H20 + energy
what are enzymes?
biological catalyst
alters RATE of reaction, not direction or products
enzyme characteristics
primarily proteins with specific structures and active sites formed when the protein folds into its three dimensional shape
why highly ordered active site
makes for enzyme specificity, only bind specific substrates
coenzyme
small molecule that participates in a rxn by donating or accepting a chemical group (a pseudosubstrate)
it is changed and used up in the rxn
enzyme cofactor
small molecules that are not changed in the enzymatic rxn, often metal ions
metal ions as cofactors
may change oxidation state and be rapidly recycled to active form
prosthetic group
tightly bound coenzyme or cofactor
six classifications of enzymes
oxidoreductases transferases hydrolases lysases ... ...
review of reaction equilibria
S P with associated delta-G
Keq of enzyme rxn
[product]/[substrate] at equilibrium
[enzyme] is not included since not consumed
transition state theory
suggests a high energy state between substrate and product with equal probability to go forward or backward
exists for time on the order of a molecular vibration, about 10^-13 second
how enzyme “works”
lowers energy of transition state between substrate and product
energetics of enzyme rxn coordinate
delta-G for rxn is unchanged
delta-G-cat much less than delta-G-uncat, i.e., transition state energy is much lower
velocity of rxn
V = k[S]
where k=(kT/h)exp(-delta-G**/RT)
k in kT term is Boltzmann constant
lock and key model of enzyme substrate rxn
actually stabilizes substrate, makes it harder to get to transition state
induced fit
stabilize the transition state, lowers the delta-G**
effective concentration
effect of an enzyme to bring substrate constituents into proximity, increasing the probability of a rxn between the substrates
alcohol dehydrogenase cofactor
zn
interacts directly with OH group in alcohol
steps to measure enzymatic parameters
1) measure initial velocity, V0, at one [S]
2) repeat your measurement at many [S]
3) create V0 vs [S] plot
enzyme velocity plot characteristics
initial plot is linear in [S]
speed levels off to reach Vmax
read Km from 1/2 Vmax
Michaelis-Menten equation
relation between Km to k-1, k1, and k2
Km = (k-1 + k2)/k1
where k1 is E+S ES
and k2 is ES E+P
Kd
enzyme binding affinity
Km
The substrate concentration at which enzyme velocity is half the maximum
Kcat
describes the rate limiting step of any enzyme catalized rxn
kcat = k2 if product release is rate limiting
turnover number Kcat
number of substrate molecules per enzyme per second that are turned into product
enzyme comparison metric
Kcat/Km
larger means more efficient processing
two classes of enzyme inhibitors
reversible and irreversible
irreversible enzyme inhibition
covalent bonding, destroy active site activity
competitive inhibition
reversibly bind to enzyme
uncompetitive inhibition
bind only to ES complex
Lineweaver-Burk plot
double reciprocal plot of
1/V0 vs 1/[S]
Lineweaver-Burk relation
1/V0 = Km/Vmax x 1/[S] + 1/Vmax
Lineweaver-Burk characteristics
Slope = Km/Vmax
competitive inhibitor
competes with substrate to bind the enzyme active site
competitive inhibitor characteristics
Kcat is unaffected while the apparent Km increases as [I] increases
Ritonavir
competitively inhibits HIV protease
Methotrexate
common cancer drug, competitively inhibits dihydrofolate reductase, a key enzyme in nucleotide biosyntheses
uncompetitive inhibitor characteristics
bind to a different site from the substrate active site
only binds to ES complex, prevents forward rxn
mixed inhibitors
bind at a different site from active site, but can bind to E or ES
penicillin
irreversible inhibitor that prevents synthesis of peptidoglycans, an integral part of bacterial cell wall
mechanisms of enzyme regulation
allosteric regulation
covalent modification of the enyme
binding of another regulatory protein
proteolytic cleavage of the enzyme to activate it
when to affect sequence of enzyme processes
earlier is better, get the first step blocked
what to use to block enzyme process
end product is good, abundance => shut down production
enzyme regulation via protoenzymes
zymogens -> enzyme via post-translational modification such as cleavage (think chymotrypsin)
