Week 6: Enzymes Flashcards
What is enzyme rate determined by?
Reaction intermediate states.
What is an enzymes main function?
Lower activation energy
What is key to enzyme specificity?
Complementarity! Van der Waals, charge-charge + hydrogen bonding.
Describe enzyme substrate cycle
Free enzyme + substrate -> ES -> EX (double dagger) - enzyme-transition state complex -> EP (enzyme-product complex) -> product + free enzyme-> starts again
What is the lock and key model?
explains specificity but not catalysis
What is induced fit model?
Induced fit model - enzyme active site and substrate undergo conformational changes - forces substrate to adopt conformation that destabilizes the enzyme-substrate complex and stabilizes the enzyme-transition state complex.
How do enzymes minimize deltaGcat(double dagger) - free energy of catalysis from ES to EX
They stabilize EX and destabilize ES
What are the 5 ways enzymes achieve rate acceleration?
Optimizing proximity and orientation of substrates, substrate and active site distortion, electrostatic catalysis, metal ion catalysis, general acid/base catalysis
Describe Optimizing Proximity and Orientation of Substrates.
Facilitation of orienting molecules for easier reactions
Describe substrate and active site distortion
Stabilize ES and transition state. Distorting substrate bonds for reactions.
Describe electrostatic catalysis
Fixed charges (ie. on backbone) in the enzyme active site are effective at stabilizing charges in TS.
Describe metal ion catalysis (2 ways - see images)
- Metal Ions as agents of electrostatic catalysis
2. Metal ions as source of OH at neutral pH (acid/base catalysis)
Describe GABC
Base catalyst - any base except hydroxide ion. Acid catalyst - any acid except H3O
GABC in enzymes use ionizable side chains ie. Glu, Asp, His
Describe histidine GBC (see images)
Imidazole side chain forms bond with water which forms a bond with a resonance stabilized carbonyl group which causes the water to attach - leaving its H and donating its OH- to the leaving group
What do proteases do?
hydrolyze peptide bonds
What are 3 types of proteases?
Serine, acid and metalloproteases
Describe how serine proteases work.
Via covalent catalysis - used by many proteases to hydrolyze peptide bonds in protein substrates. Serine proteases - use deprotonated serine R-O- as nucleophile - attacks peptide bonds - convert to ester (acyl enzyme intermediate) - then attacked by water.
Serine proteases have highly reactive serine @ active site.
What is the catalytic triad
Catalytic triad - Ser 195, His 57, Asp 102
What are three types of serine proteases
Chymotrypsin (cuts after Trp, Tyr, Phe), Trypsin (cuts after Lys, Arg) and Elastase (cuts after Ala and Ser)
Describe the process of the catalytic triad generating a good nucleophile (see images)
The negative charge on Asp 102 orients His 57 correctly and stabilizes protonated state. His 57 acts as general base - deprotonating Ser 195 - forming alkoxide. Deprotonated Ser 195 forms covalent bond with substrate.
Describe the catalytic cycle of chymotrypsin
Ser 195 - covalent catalysis
His 57 aided by Asp 102 - GABC - His 57 2x base 2x acid
Oxyanion hole - electrostatic catalysis - stabilizes two tetrahedral oxyanion intermediates
Step 1: Polypeptide substrate binds noncovalently in enzyme active site. Ser attacks the electrophilic amide C.
Step 2: The resulting tetrahedral oxyanion is stabilized by H-bonding interactions with oxyanion hole.
Step 3: Collapse of the tetrahedral intermediate and proton transfer from His lead to cleavage of C-N bond - the N-terminal peptide is bound through acyl linkage to serine. C-terminal fragment is cleaved.
Step 4: Water binds to the active site and attacks the acyl ester carbonyl.
Step 5: The resulting tetrahedral oxyanion intermediate is stabilized via enthalpic interactions with oxyanion hole
Step 6: The second peptide fragment (N-terminal fragment) is released and the enzyme returns to its initial state.
What is two examples of acid proteases
Pepsin = non-specific, HIV protease - specific (HIV antiretrovirals are inhibitors of HIV protease active site).
Describe the acid protease mechanism
Two asp molecules - one begins as deprotonated. The protonated one acts as an electrostatic catalyst and the deprotonated as GABC
Step 1: An oxygen on the deprotonated Asp attacks a hydrogen on water - keeping it and leaving OH-
Step 2: OH- attacks the carboxyl group which relocates its electrons to the double bonded oxygen (single bond, 3 electron pairs)
Step 3: The oxygen then adds its electrons back to make a double bond and the peptide bond is cleaved, the H on the now protonated Asp is added to the N terminus and it is now deprotonated
Describe a zinc metalloprotease - carboxypeptidase A
Zn2+ can generate OH-from water in enzyme active site at physiological pH. This can be used in hydrolysis reactions, as in digestive protease carboxypeptidase A. The chemical environment in Carboxypeptidase A further inhances zincs ability to generate bound hydroxide - higher pH.
Carboxypeptidase A is a C-terminal exopeptidase - cuts peptide bonds starting at C-terminal with addition of water.
Describe the mechanism of carboxypeptidase A
Has a positive Arg145 that holds the C-terminus in place. 2 charged arginine + deprotonated (charged) glutamic acid + zinc (II)
Glu → acting as general base
Step 1: A Zn is bound by His 69, 196 and Glu 72 - binds OH which then attacks the carboxyl which sends its electrons to the double bonded oxygen. This negative charge is stabilized by a positive Arg 127
Step 2: The electrons shift back into a double bond, and the peptide bond is cleaved - relocates its electrons to a protonated Glu 270.
Step 3: water is removed and Glu 270 becomes deprotonated.. A H then joins the ZnOH and this releases the C terminus.
Where else are acid protease mechanisms used?
in other hydrolases ie. esterases, phosphatases, amidases
What are 3 strategies for regulation of enzyme activity?
- Irreversible covalent activation (zymogens)
- Irreversible covalent inhibition
- Reversible covalent modification of the enzyme
With what type of enzyme does irreversible covalent activation usually occur?
Describe an example with chymotrypsinogen
with proteases - synthesized in inactive form to prevent degradation of cell that expresses them.
The inactive precursor is zymogen - ie. chymotrypsinogen.
The enzyme is activated by proteolysis at specific peptide bonds
This is irreversible as once these peptide bonds have been cut, they cannot be reformed.
In chymotrypsinogen - substrate binding site is blocked. On activation by proteolysis the substrate channel is accessible. The processing sites are not near the catalytic triad - these residues are activate but cannot attack substrates until binding site is cleared.
Describe Irreversible covalent inhibition
Serine protease inhibitors (serpins) - class of covalent inhibitor proteins ie. antitrypsin. Serpin - binds noncovalently to active site Antitrypsin binds to trypsin to form trypsin-antitrypsin complex
Describe Reversible covalent modification of the enzyme
Specific amino acid residues with alcohol side chains on enzymes can be reversible phosphorylated (ATP -> kinase -> ADP) or dephosphorylated (H2O -> phosphatase -> phosphate)
Phosphotyrosine and phosphothreonine move towards nearby arginine residues and in doing so, they flip away from active site and make it available to substrates. Phosphatases can restore enzyme to original state
ie. MAPK inactive (ATP -> MAPK kinase -> ADP) MAPK Active (H2O -> MAPK phosphatase -> phosphate) - MAPK inactive
What is velocity? Instantaneous rate?
Change in speed over change in time - or change in product concentration over change in time = concentration. Instantaneous rate is the tangent
What is the velocity for a first order reaction?
v0 = k[S]0 - rate is proportional to [S] - units sec-1
What is the velocity for a zero order reaction?
Rate is constant: v = k - units = concentration/time
What is saturation?
For a typical enzyme-catalyzed reaction, the rate “maxes out” - exhibits saturation - this dependence of rate on [substrate] is described by hyperbolic function. At low substrate - reaction is 1st order, high substrate - reaction is zero-order
What is the Michaelis-Menten equation?
hyperbolic function: y = ax/(b+x) where a and b are constant. For small values of x - b+x is nearly equal to b: y = ax/b
For large values of x, b+x = x equation is y = a - horizontal line - zero order reaction.
Equation: v = Vmax[S]/Km+[S]
Where [S] is initial substrate concentration, vmax = max rate, Km = Michaelis constant
What are the assumptions of the Michaelis-Menten equation?
- ES complex is intermediate
- Concentration of ES remains constant - steady-state assumption
- Initial rates of reaction are considered, the contribution of the back reaction is neglected
- Total [enzyme] = [free enzyme] + [ES complex]
Describe E + S -(k1)-> ES -kcat-> E + P
kcat and Km are properties of enzyme catalyzed reaction - depend on enzyme and substrate
kcat = number of catalytic cycles per second for enzyme molecule = Vmax/[E]t
Vmax depends on enzyme concentration. kcat is independent of enzyme concentration - inherent property of enzyme
Km is derived from rate constants - hase units of concentration. Lower the Km the lower the concentration fo substrate needed to reach rate saturation. It is substrate concentration when v = 1/2 vmax
[S] = Km when v = 1/2vmax
The smaller the Km the lower the [S] required to achieve rate saturation
How do you get Km and Vmax from a linear equation?
plot 1/v against 1/[S] - Lineweaver-Burk Plot (double-reciprocal plot).
The x-int is -1/Km, the y-int is 1/Vmax and the slope is Km/Vmax
Can kcat and Km tell how effective an enzyme is?
kcat and Km alone are not enough to tell how effective enzyme is.
Together, can rate catalytic power of enzyme: enzyme efficiency = kcat/Km - second order rate constant with units /Msec - higher the better - dependent on substrate
When are enzyme efficiencies used?
- To determine substrate preference of enzyme 2. To determine how mutations affect enzyme activity
