Exam 2: Enzymes Flashcards
What is catalysis
the lowering of activation energy of a reaction causing it to speed up.
What are the characteristics of an enzyme (6)
- Increases the rate of a reaction
- Can catalyze both a forward and backward reaction.
- They can be present in low concentrations
- Transition state of reacting substrates bound in enzyme active site
- they obey the laws of thermodynamics
- They can be regulated.
Do not effect equilibrium
highly specific to substrates
What is a transition state
structural form of substrate with highest energy state along the complete rxn pathway
What is the activation energy
the difference between the transition state and reactants
What is an active site
unique binding site on the enzyme for a substrate to bind, a cleft or crevice in enzyme.
How does the active site help with catalysis
the R groups of the AAs in the active site interact with the transition state helping speed up rxn. The shape of the active site can force a substrate to adapt its conformation to be similar to the transition state
What is the difference between catalysis in vitro and in vivo
the crowding effects in a cell make the activation energy levels lower, rxn is faster
Explain enzyme specificity
before thought of as lock and key. Now induced fit: substrate bonds noncovalently to active site, changing conformation of the enzyme to make it perfectly fit to substrate.
What` are the 2 classes of enzyme cofactors
metal ions: Mg, Zn, Co
coenzymes: small organic molecules usually not covalently attached.
What are the 6 types of enzymes, how are they classified
classified by type of rxn.
oxidoreductase: oxidation reduction rxns
transferase: transfers a group from a mol to another
hydrolase: cleaves a bond using H2O
lyase: removes or add groups to a double bond
ligase: forms a new bond
isomerase: rearrangement
What groups does transferase often move?
amino, carbonyl, carboxyl, methyl, phosphate, acetyl
Name an example of an oxidoreductase enzyme
alcohol dehydrogenase
Name an example of a transferase enzyme
hexokinase
Name an example of a hydrolase enzyme
chymotrypsin
Name an example of a lyase enzyme
pyruvate decarboxylase
Name an example of an isomerase enzyme
alanine racemase
Name an example of a ligase enzyme
pyruvate carboxylase
What is km
michaelis menten constant. measures enzyme affinity for substrate. [substrate at 1/2 Vmax]
What does a large and small km mean
large: enzyme has low affinity
small: enzyme has high affinity
What is kcat
the # of substrate mols converted to product per unit of time by an enzyme mol under saturating conditions (steady state). vmax/[E total]
What does a large and small kcat mean
large: more product per unit of time
small: less product per unit of time
what kind of kcat and km values does the ideal enzyme have
small km and large kcat
What is the formula for the specificity constant? what does it represent
the overall efficiency of the enzyme
kcat/km
What are the values for the y and x axis on the michaelis menten plot
x: [S]
Y: Vo
What are the values for the y and x axis on the Lineweaver Burke Plot
X: 1/Vo
Y: 1/[S]
What is the formula for the slope of a lineweaver burke plot
slope=km/Vmax
What is the x and y intercept on the lineweaver burke plot
y intercept: 1/Vmax
x intercept: -1/km
What are the 2 kinds of multisubstrate rxns
- sequential: can be ordered ( enzyme binds to substrate A then B must go next, 1 specific order) or random ( 2 substrates but can be added in any order, rate can change depending on order)
- Double displacement: ping pong, enzymes can have altered conformations, rxns happen one at a time but they rely on each other
What are the 2 types of enzyme inhibitors
irreversible: less common naturally, covalent bonding enzyme permanently inhibited
reversible: inhibitor binds reversibly to enzyme usually noncovalent bonds
What are the 3 subtypes of reversible enzyme inhibitors
competitive: inhibitor competes with S for same binding site
non competitive: inhibitor binds to E at a site other than the active site
uncompetitive: inhibitor binds to ES complex only
How can a competitive inhibitor be overcome
too much substrate must be added to overwhelm it, this is the only type of inhibitor that can be overcome
would a competitive inhibitor rather have a large or small alpha?
x intercept with competitive inhibitor= -1/alpha km
larger alpha makes it more effective change to km
What is an example of a competitive inhibitor
Fumerate is a competitive inhibitor to succinate (S), because it has a similar structure (analog)
What is the difference between pure and mixed noncompetitive inhibitors
pure: vmax goes down, km no change
mixed: vmax goes down, km can go up or down
What are enzyme mechanisms
intermediate species that exist only briefly (long enough to isolate experimentally). can be a carbocation( e- deficient C+), carbanion (unshared e- C- , acts as nucleophile) or free radical ( atom with single unpaired e- very reactive)
what is an example of an enzyme mechanism. Describe rxn by answering Q in notebook. What is the enzyme, substrate, and product
triosphosphate isomerase TPI
enzyme: TPI
substrate: DHAP
product glyceraldehyde 3-phosphate
How does TPI catalyze the rxn
using Glu and His in its active site, it interacts with the S and transition state in a 2 step rxn.
What are the 5 types of catalytic mechanisms
preferential state binding proximity and orientation effects electrostatic interactions acid base catalysis covalent catalysis
Describe preferential state binding
E active site has a stronger affinity for the transition state than substrate or product. TS fits better in active site than original S
Describe proximity and orientation effects
2 substrates are in close proximity and they are oriented into active site so that catalysis can occur
describe electrostatic interactions as an enzyme catalytic mechanism
non covalent bonds in active site between substrate and enzyme R groups. Lowers the AE
Describe acid catalysis
R group of E in active site donates H+
Describe base catalysis
R group of E active site withdraws or takes H+ from S or another R group
What are good R groups for acid/base catalysis
His, Asp, Glu, Tys, Cys, Lys because they have COOH or N+H3
Describe covalent catalysis.
S covalently binds to R group in active site in a non permanent way
Answer match for types of enzyme catalysis in notebook
match image to type of catalysis, circle the covalent bond
What are enzyme cofactors
non AAs of active site that help with catalysis like metal ions and organic mols
What kinds of metal ions are cofactors. What kind of effects are present in the rxn. Identify parts of rxn in notebook
Zn, Fe and Cu
proximity orientation effect, noncovalent attraction, An acts as base
What kind of organic mols are cofactors
usually vitamins, water soluble, lipid soluble or vitamin like molecules
How does temperature effect enzyme activity
increase in T causes increase in molecular motion causes increase in enzyme activity. Very high temp causes denaturation ie lowers E activity or stops it. Range depends on R groups in enzyme
How does pH effect enzyme activity
affects acid base catalysis and ionization state
Where are pepsin and chymotrypsin present, what does this have to do with their preferred ph ranges?
pepsin: in the stomach ideal pH is 2
chymotrypsin: in the pancreas ideal pH is 8
What is chymotrypsin? what does it do
very large enzyme, in serine protease family, catalysis the hydrolysis of peptide bond adjacent to an aromatic R group. Contains catalytic triad in active site: Asp, His, Ser
Describe the 6 steps of the action of chymotrypsin, use image in book pg 224
a) the catalytic triad creates a temporary oxyanion hole (unoccupied), hydroxyl O of Ser 195 nucleophilically attacks the carbonyl C of the substrate (covalent catalysis), Asp acid 102 polarizes the His 57 (electrostatic catalysis),
b) His acts as general base to abstract proton from Ser in active site. Ser covalently attached to substrate. Intermediate 1 preferentially binds to enzyme
c) His acts in acid catalysis, protonating S breaking peptide bond causing amine terminus to leave.
d) H2O enters active site where N terminus has left, acts as nucleophile
e) His does base catalysis on H2O in active site, making H2O a good nu and an oxyanion intermediate forms
f) decomposition is facilitated by acid catalysis of His, regenerated active site and product are released
Explain the relationship between alcohol dehydrogenase and Zn
Can’t be active without zinc, has 2 zincs attached to each subunit (2-4 subunits). 1 zinc ion is in the active site the other not in the active site has a structural function. The electrostatic effect that Zn causes stabilizes the transition state
Describe the rxn of alcohol dehydrogenase, use image in notebook, write the rxn, who is reducing and who is oxdizing? What does alcohol dehydrogenase do in this rxn
oxidizes alcohols mostly ethanol to aldehydes or sometimes to ketones.
a) alcohol dehydrogenase has 2 Cys, His and Zn in its active site, the substrate ethanol is binding to Zn as the alcoholate anion, water is in the active site.
c) NAD is binding to the active site causing conformation changes and expelling H2O allowing ethanol in to bind to the zinc (stabilizes transition state)
NAD+ accepts a hydride ion from the substrate and the aldehyde product is formed.
What are the 4 types of enzyme regulation?
genetic
covalent modification (rapid)
allosteric regulation
basic compartmentalization: physical separation of enzymes and substrates (organelles)
What are the 2 types of covalent modification in enzyme regulation
reversible: covalent bond of a small organic mol (chemical group) to an enzyme, conformation of enzyme changes causing either lowering or increase in activity of the enzyme
irreversible: some enzymes are produced and stored as inactive precursors (proenzyme/ zymogen). Activates when zymogen is cut causing conformation change irreversibly. can only be stopped by degradation
Give examples of both kinds of covalent modification as enzyme regulation
reversible: phosphorylation ( kinase) and dephosphorylation (phosphatases)
irreversible: activation of chymotrypsinogen
Explain the process of the activation of chymotrypsinogen
Chymotrypsinogen is a inactive (zymogen), trypsin attaches to it between Arg15 and ile16 and causes a conformation change turning it into pi chymotrypsin (semi active, 2 separate pp chains).
Cymotrypsin cleaves to each mol causing them to activate each other and the result is 3 pp active chains of alpha chymotrypsin.
What is allosteric regulation?
fast, effector mols reversibly bind (noncovalently) to a site other than the active site. Binding causes conformation change of enzyme to either activate or inhibit it
Explain how allosteric regulation inhibits and activates an enzyme
inhibition: the allosteric inhibitor binds to an alternative site on the enzyme and distorts the active site making it so that the substrate can no longer enter it.
activation: an enzyme with a distorted active site attaches to an allosteric activator, causing the active site to no longer be distorted and the substrate may enter it.
What are other ways to say allosteric inhibitor
effector, sometimes also ligand
How do allosteric inhibitors and activators affect Vmax and km
inhibitors: lower vmax, increase Km
activators: increase vmax, decrease km
Explain what feedback inhibition is
can be a type of allosteric inhibition where the product of a pathway inhibits the activity of an enzyme at or near the beginning of the pathway.
Give an example of feedback inhibition
ATCase: In the reaction of carbonyl phosphate + aspartate –> CTP, ATCase can initite the rxn and can also stop the rxn when CTP is no longer needed. ATCase binds to CTP and allosterically inhibits the rxn pathway. when [ATP] is higher than CTP, ATCase acts as an activator, When [ATP] is lower than CTP, ATCase acts as an inhibitor
What are the major coenzymes? Describe the function of each
- High energy potential: activate metabolic intermediates and or serve as phosphate donors or as carriers for small molecules.
- Electron or hydrogen transfer: causes a set of reactions where C atoms are transferred in various oxidation states between substrates
- Group transfer: transfer of groups like aldehyde acyl and amino groups
What properties of transition metals make them useful as enzyme cofactors
- They can bind to functional groups or can be components of prosthetic groups like iron in heme.
- provide high [] of positive charge that is good for binding small molecules
- they are lewis acids so they make good electrophiles
- d shell lets them interact with 2+ ligands helping orient the substrate within the active site
- have two or more valence states, lets them mediate oxi-red rxns by reversibly gaining or losing e-
Draw the structure of the amino acids whose side chains most commonly participate in catalytic mechanisms when they occur in the active sites of enzymes.
His (imidazole ring), Asp, Glu (carboxylate), Tyr (hydroxyl), Cys (sulfhydryl) , Lys (amine)
All functional groups can act as both proton donors (acids) or proton acceptors (bases)
Describe compartmentation within eukaryotic cells. What problems does this phenomenon solve for living organisms
Compartmentation within eukaryotic cells is the physical separation of enzymes by a membrane or by attachment of enzymes to membranes or cytoskeletal filaments.
- prevents competing rxns - allows separate regulation of rxns - reduces diffusion barriers by putting enzymes and metabolites near each other - provides specialized rxn conditions (pH, or temp). - protects other cellular components from toxic rxn products.
Explain the difference between the energy of a reaction and the energy of activation
The energy of activation is the minimum energy required to cause a chemical reaction. The energy of a reaction is the total energy used during a reaction.
An enzyme catalyzes a reaction with a Km of 4.31 and a Vmax of 3.45mM/s. Calculate the reaction velocity vo
at a substrate concentration of 8mM. (formula in given info)
2.24 M/s
Below is a mechaelis menton plot for an enzyme catalyzed rxn. The rxn is a first order. Why is the shape hyperbolic? why the the rate level off at higher concentrations of substrate?
The rate levels off because the enzyme has become saturates with substrate. This means it has reaches its maximum rate of catalysis
Catalase has a Km of 25 mM and a kcat of 4.0x10^7sec-1 with Hydrogen Peroxide as a substrate.
Carbonic Anhydrase, has a Km of 26mM and a kcat of 4.0x10^5 sec-1 with the same substrate
a. ) Which enzyme has a higher affinity for the substrate Catalase or Carbonic Anhydrase?
b. ) Which enzyme is more efficient at converting substrate to product?
c. ) Calculate the specificity constant for each enzyme. Type the values in the third response box.
d. ) Looking at the specificity constants you calculated for each enzyme, which enzyme is more efficient overall?
a) catalase
b) catalase
c) catalase: 1.6 x 10^6, carbonic anhydrase: 1.5 x 10^4
d) catalase
