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64 Cards in this Set

  • Front
  • Back
What reaction does carbonic anhydrase catalyze?
Hydrolysis of carbon dioxide
Where does trypsin cleave?
Hydrolyzes a peptide bond after lysine or arginine
What is a suicide inhibitor?
Most specific irreversible inhibition:
Inhibitor binds to the enzyme as a substrate and is initially processed by normal catalytic mechanism -- generates a chemically reactive intermediate that inactivates the enzyme through covalent modification
What is a group-specific reagent?
Irreversible inhibitor:
Reacts with specific side chains of amino acids.
What is an affinity label?
Irreversible inhibitor:
structurally similar to substrate for enzyme -- covalently binds to active-site reside. More specific than group-specific reagents.
ΔG = ?
ΔG = ΔG⁰ + RT ln[C][D]/[A][B]
What does ΔG tell us?
Whether a reaction will occur spontaneously. negative = yes, positive = no, zero = equilibrium
Three pieces of evidence that tells us enzymes and substrates form a complex
1. If you increase [S], reaction rate increases. Uncatalyzed reactions do not show this "saturation" effect

2. X-ray crystallography

3. Spectroscopic characteristics (different for enzyme and substrate separately)
Five common features of enzymes:
1. active site is a cleft/crevice
2. active site takes up relatively small part of total enzyme volume
3. active sites are unique microenvironments
4. substrates bind to enzymes by multiple weak attractions
5. specifity of binding depends on arrangment of atoms in active site
What is the most stable interaction, between enzyme and what?
enzyme + transition state
Does ΔG correlate with rate?
No. It is related to the equilibrium constant of a rxn, therfore, the concentrations of reactants and products.
What is the steady-state assumption?
That enzyme-substrate complex is being formed and broken down at the same rate, so overall [ES] is constant
What is a first order reaction?
A reaction that is directly proportionate to reactant concentration
What is a second order reaction?
When there are two reactants.
What is a pseudo-first order reaction?
If one reactant is present in excess and the other is present at low concentration, then reaction rate will only depend on concentration of low reactant
What is the M-M equation for Km?
Km = k₋₁ + k₂ / k₁
What is the M-M equation for [ES]?
[ES] = [E][S] / Km
What is the M-M equation for Vmax?
Vmax = k₂[E]t
What is the M-M equation for V₀?
V₀ = Vmax* [S]/[S]+Km
What does Vmax mean?
the turnover number of an enzyme -- the number of substrate molecules converted to product by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate.
What is the oxyanion hole?
The nitrogen of histidine interacts with the negatively charged oxygen of the substrate, stabilizing its negative charge.
What are the five key features of an enzyme's active site?
1. 3D cleft/crevice
2. Takes up small % of total enzyme volume
3. Unique microenvironment
4. Binds to substrate by many weak bonds
5. Specificity depends on arrangement of atoms
What does a large Ki mean? What does a small Ki mean?
Large Ki = weak inhibition because more of the inhibitor is free

Small Ki = strong inhibition because more of the inhibitor is bound to the enzyme
What did Linus Pauling propose?
Compounds resembling transition states make good inhibitors
What does adenylate kinase do?
It catalyzes the transfer of terminal phosphate group from ATP onto an NMP
How does an aspartyl protease work?
COO- (deprotonated aspartic acid) activates a water molecule, making it a potent nucleophile which attacks the carbonyl group of the peptide. A second aspartic acid (protonated) polarizes the peptide carbonyl group so that it is more susceptible to attack.
How doe metalloproteases work?
Zn2+ stablizes a water molecule. A base (-B:) attacks the water molecule, removes a Hydrogen. Activated water now attacks carbonyl.
What does carbonic anhydrase do?
Catalyzes the reaction of C02 + H2O into carbonic acid into bicarbonate ion.

1. Zinc ion facilitates the release of a proton from water, which generates an OH- (abstracted by Histidine)
2. CO2 substrate binds to enzyme's active site and is positioned to react with OH-
3. OH- attacks CO2, converting it into HCO3-
4. Catalytic site is regenerated with the release of HCO3- and bnding of another molecule of water.
What is the histidine proton shuffle?
Histidine abstracts a proton from water bound to zinc, generating a nucleophilic hydroxide ion and a protonated histidine. The proton is then removed by a buffer, regenerating histidine.
What is a DNA molecule called that contains a recognition sequence?
cognate DNA
What is a restriction-modification system?
Host cell produces corresponding methylase that marks the host DNA at the appropriate methylation site for every endonuclease that it targets, because host endonuclease will not cleave DNA if its recognition sequence is methylated.
Where do endonucleases cleave?
Catalyze hydrolysis of phosphodiester backbone of DNA: bond between 3' oxygen atom and phosphate group.
Endonuclease Mechanism to Retain Stereochemistry:
1. Nucleophile attacks phosphoryl group to form covalent intermediate.

2. Intermediate is hydrolyzed to get final product.

* Two displacement rxns take place at PO4 atom -- stereochemistry is inverted and then inverted again to retain original stereochemistry
Endonuclease Mechanism to Reverse Stereochemistry:
Activated water molecule attacks phosphorous molecule directly
- single displacement reaction
* stereochemistry is inverted after cleavage
What is the role of magnesium in endonucleases?
Helps to position and activate water molecule for attack of phosphorous atom.
What is the correct mechanism by which endonucleses cleave DNA?
Direct hydrolysis: attack by water molecule.
* Determined by EcoRV: replacing oxygen with sulfer, saw no inversion of stereochemistry
How do restriction enzymes know where to cleave?
They can bind to any sequence (run along the sequence), but are able to create a bend/kink in the correct sequence.
- The bend allows positioning of sequence close enough to active Asp residue to bind to Mg2+
Horizontal gene transfer
The passing between species of pieces of DNA (such as plasmids) that provide selective advantage in a particular environment.

When two species share this piece of DNA for a particular enzyme but are not closely related, it is likely that they got the gene more recently than time of evolutionary divergence.
NMP binding domain
Central B-sheet surrounded by a-helices: loop between 1st B- and a-
* P-Loop: interacts with phosphoryl group on both nucleotide
4 Steps of carbonic anhydrase:
1. Water deprotonation: generates OH-
2. Carbon dioxide binding
3. Nucleophilc attack of hydroxide on CO2
4. Displacement of bicarbonate ion by water
How does NMP Kinase prevent transfer of phsophoryl group to water?
Induced fit. Also, metal ion forms complex with substrate (not enzyme) - does so with NMP not water. Also, Mg2+ coordinates with water, keeping it away from the active site.
What is the role of Mg2+ in endonucleases?
The magnesium ion helps activate a water molecule and position it so that it can attack the phosphorous atom.
What is the role of the metal ion in NMP Kinase?
Metal is not a component of the enzyme -- it binds to the substrate making a mtal-ion-nucleotide which becomes the substrate that the enzyme bind to.

Magnesium + Phosphoryl group oxygen: holds nucleotide in well-defined conformation so that enzyme can bind.
How does NMP kinase work?
1. Substrate binding induces large structural changes in the kinase:

2. P-Loop closes down on polyphosphate chain: interacts w/ B-phosphoryl group

3. Movement of P-Loop brings down domain of enzyme
- Forms "lid" over bound nucleotide

4. Lid holds ATP in position
- Gamma-phosphoryl group is positioned next to the binding site for next NMP to bind

5. Binding of NMP induces more changes

** This only happens when both donor and acceptor are bound, prevents transfer of PO4 to water.
What type of catalysis occurs in NMP kinase?
Catalysis by approximation
- The two substrates are held close together only when both donor and acceptor are bound. Prevents transfer of PO4 to water.
What is the name of an inactive precursor enzyme?
Zymogen / Proenzyme
What are the components of ATCase?
2* c₃ trimers
3* r₂ dimers
= 12 subunits
What stabilizes the R chains of ATCase?
zinc ion bound to 4 cystein residues
How do we know that ATCase has distinct regulatory and catalytic subunits?
Treatment with mercurial compound separates the subunits.
* Further - ultracentrifugation and ion exchange chromatography, because the groups differ in size/charge
What do the different peaks correlate with in the sedimentation separation of subunits of ATCase?
large peak: native ATCase
first small peak: r₂
second small peak: c₃
Which ATCase subunit is which?
Larger: catalytic subunit
* displays catalytic activity, does not respond to CTP
* does not display sigmoidal kinetics

Smaller: regulatory
* can bind to CTP, but has no catalytic activity
How does mercury tell us that there are two different subunits in ATCase?
Mercurial compound is able to dissociate C and R subunits because it binds strongly to Cystein residue
- displaces zinc: destabilizes R-subunit domain
What are the two forms of ATCase?
T - no substrate - low affinity - low catalytic activity

R- bound substrate - higher binding affinity - binding to one site increases liklihood that enzyme shifts to R
Which model of cooperativity does ATCase display?
Concerted Model:
- all or none: entire enzyme is either T or R state.

(not Sequential model)
How does CTP work?
It shifts equilibrium to the T-STATE
- decreases binding affinity for substrate (and therefore total enzyme activity)
- binding of CTP makes it more difficult to convert to R state: decreases potential for cooperativity

* more substrate required to give faster rxn rate
What is CTP?
Product of ACTase reaction, serves to inhibit it.
- when bound, enzyme is in T-state
- interacts with regulatory chain, not catalytic chain
Homotropic effect
The effect of substrate on allosteric enzyme
Heterotropic effect
effects of non-substrate molecules on allosteric enzymes (regulators)
How to ATP and CTP interact in ATCase?
ATP competes with CTP regulatory sites to INCREASE R-STATE
* Allosteric modulator of ATCase, increses it!
* High ATP prevents CTP from binding to inhibit
Differ in amino acid sequence but catalyze the same reaction
- display different kinetic characteristics such as Km; or respond to different regulatory moleucles

** Distinguishable from one another by isoelectric focusing**
Lactate Dehydrogenase
goal: to replenish NAD+ levels in the cell.
- important in fermentation / low O2 in cytoplasm
Strength of ES binding?
-13 - -50kJ/mol
Line-Weaver Burke Plot Equation
1/V = Km/Vmax*S + 1/Vmax
Michaelis-Menten Equation
V = Vmax[S]/[S]+Km