Enzyme Kinetics Flashcards
Enzymes
Substances that catalyze reactions without changing themselves (may change during reaction, but always return to their original state)
Work by lowering activation energy: stabilize transition state
Speed up rate of reaction
Specificity of an enzyme
Determined by interaction with substrate
Wide range of specificity: some cleave any peptide bond (papain), whereas some cleave only certain bonds (thrombin: cuts bonds between Arg and Gly)
Cofactor
Form complex with enzyme, allowing it to function
Holoenzyme
Enzyme and cofactor
Apoenzyme
Enzyme without cofactor
Prosthetic
A cofactor that is tightly bound to its enzyme
Cosubstrate
A cofactor that is loosely bound to its enzyme
Transition state
Structure between substrate and products
Has highest energy and is least stable
Enzymes bind to these and stabilize them
How the best drugs work
Best drugs inhibit substrate from reaching transition state: they add an inhibitor that mimics the transition state
The rate of product formation is proportional to…
Transition state formation
Why enzymes are necessary
Normally, to clear the Ea barrier, large amounts of kinetic energy that come from an increase of temperature are needed
The body can’t tolerate large increases in heat, so enzymes are needed to lower the Ea
Maximal velocity
Point at which all active sites on enzyme are occupied
Never actually reached: plot of [S] vs. reaction velocity (V not) is asymptotic
How to determine the active site of an enzyme
- Co-crystallography: crystallize enzyme and substrate to see binding
- See what enzyme reacts with
- Induce mutations into the enzyme and see their effect on binding
Spectral evidence for enzyme-substrate binding
Spectral changes are observed when substrate is added to enzyme, especially with color-producing reactions
Active site
Place where substrate binds to enzyme
Features of active site
3-D: folding dictates
Small part of enzyme (easily manipulated)
Specific amino acids dictate what can bind
Multiple non-covalent bonds are at work
Induced fit
Enzyme and substrate change each other to maximize binding
What enzymes can affect
Only the amount of energy needed to initiate
Never delta G
Exergonic reaction
Change in delta G is negative
Spontaneous reaction
Equilibrium
Delta G equals 0
Equal substrate and product formation
Endergonic reaction
Change in delta G is positive
Input of free energy is needed for reaction to proceed
Delta G and rate of reaction
Delta G provides no info about rate of reaction
Rate is dictated by Ea
Free energy of reaction equation
Delta G= Delta G not + RT ln ([C][D])/([A][B])
Delta G not= standard free energy change at pH 7
R= gas constant
T= absolute temp
Equilibrium constant equations
Keq= ([C][D])/([A][B]) Keq= 10^(-delta G/2.303RT)
1st order reaction equation
V=k[S]
k= constant in s-1
[S]= substrate concentration
2nd order reaction equation
V=k[S]^2 (k is in s-1)
V=k[S1][S2] (k is in M-1 s-1)
Michaelis constant equation
KM= (k-1+k2)/k1
k-1 and k2: breakdown of transition state
k1: formation of transition state
Steady state assumption
Equilibrium: equal amount going to and taking away from
Michaelis-Menton equation
V not= (Vmax * [S])/(KM + [S])
Lineweaver-Burk plot
Plotting reciprocal of MM equation can allow measurement of V not and [S]
X intercept= -1/KM
Y intercept= 1/Vmax
Slope= KM/Vmax
Only works well if data points make good line
Sequential reaction
All substrates must bind enzyme before the product is released
Can be ordered or random
Ordered sequential reaction
Substrates must bind in a certain order and be released in a certain order
Enzyme undergoes conformational change
Random sequential reaction
Doesn’t matter in what order substrates are bound and released
Enzyme either doesn’t undergo conformational change or undergoes small change
Double displacement (“ping pong”) reaction
One or more products are released before all substrates bind
Hallmark: intermediate enzyme modification (modified until all products have been formed)
Allosteric enzymes
Control from sites separate from the active site
Don’t follow MM kinetics: sigmoid rather than hyperbolic in plot of [S] vs. V not
Reversible inhibitors
Can be displaced if inhibitor binds to active site
Non-covalent bonding
Used in drugs: need to inhibit some functions, but not all
Irreversible inhibitors
Cannot be displaced
Covalent bonding
Competitive inhibitor
Reversible inhibitor
Binds to same site as substrate
Usually looks like substrate
Uncompetitive inhibitor
Reversible inhibitor
Binds to site near active site, but not active site itself
Binds when substrate is present and prevents transition to product
Noncompetitive inhibitor
Reversible inhibitor
Binds to completely different site than active site
Allosteric inhibitor: changes shape of active site, disrupting substrate binding
Reciprocal plot of competitive inhibitor
No change in Vmax KM increases (closer to 0): more substrate needed to reach 1/2 Vmax ([S]=KM)
Reciprocal plot of uncompetitive inhibitor
Vmax decreases (further from 0) KM decreases (further from 0): less substrate needed to reach 1/2 Vmax ([S]=KM)
Reciprocal plot of noncompetitive inhibitor
No change in KM: active site is still available
Smaller Vmax: substrate binding is disrupted
