enzymes Flashcards
What is Km?
Michaelis constant. concentration of substrate required for 1/2 Vmax of enzyme substrate complex
can also be an APPROXIMATE measure of substrates affinity for enzyme (if k2<
what is Vmax?
the maximum velocity of formation of the enzyme substrate complex.
Vmax=kcat[Etot]
what is kcat?
the turnover number. how many substrate molecules can one enzyme molecule covert per second.
can be approximated by rate constant of rate limiting step (if only one) or a combination of several rate constants if there is no obvious single step.
kcat=Vmax/[Etot]
what is the specificity constant?
kcat/Km
measure of the rate constant for an enzymatic reaction covering E+S to E+P. Most efficient when [S]<
if [S] is very large, what determines velocity?
Vmax. the equation can be reduced to Vo=Vmax
if [S] us very small, what determines velocity?
Vmax and Km and [S].
equation is only slightly different
V0=Vmax[S]/Km
what effect does increasing [S] have on velocity?
increased [S] increases velocity because there are more substrate molecules to interact with. There is a decrease in rate over time because the [S] is decreasing as it is being converted to P
2 substrate reactions: Sequential ordered mechanism
enzyme first forms complex with S1. the S2 can only bind to the ES1 complex, not to the E, to form the ternary complex.
Lineweaver-Burk lines intersect. (cannot easily distinguish from random)
2 substrate reactions: Ping Pong mechanism
The binding of S1 to E causes a modified E to form (E’). The E’ releases P1 and then can bind S2. The E’S2 complex then releases P2 and unmodifies the E’ to reform E.
Lineweaver-Burk: lines are parallel
2 substrate reactions: Random order mechanism
E can bind to either S1 or S2 first and then binds to the other to form ternary complex. Not sequential
Lineweaver Burk: lines intersect. (cannot easily distinguish from sequential)
What are inhibitors? What are the two types?
compounds that decrease enzyme’s activity. There are Irreversible inhibitors and Reversable inhibitors
What are irreversible inhibitors?
inhibitors that react with the enzyme and can permanently shut off one enzyme molecule. They are often powerful toxins but can also be drugs. A suicide inhibitor is only functional once, it only shuts down 1 enzyme.
What are reversible inhibitors?
inhibitors that bind to and can dissociate from the enzyme. They are often analogs of substrates/products and are used as drugs. reversible inhibitors can bind to the free enzyme and prevent substrate binding or to the enzyme-substrate complex to prevent reaction (competitive vs noncompetitive)
Competitive inhibition
competes with substate for binding by binding active site. doesn’t effect catalysis
No change in Vmax, apparent increase in Km
Lineweaver-Burk: lines intersect at the y-axis
alpha is the factor by which Km increases as [I} increases. alpha defines effectiveness of inhibitor
uncompetitive inhibition
only binds to ES complex, does not effect substrate binding. inhibits catalytic function
Decrease in Vmax, apparent decrease in Km
No change in Km/Vmax
Lineweaver-Burk: lines are parallel
alpha’ is the factor by which Km and Vmax decrease
mixed inhibition
binds enzyme with or without substrate. binds to regulatory site and inhibits both substrate binding and catalysis.
Decrease in Vmax, change in Km (increase or decrease)
Lineweaver-Burk: lines intersect left from y-axis
involves both alpha and alpha’
what are allosteric regulators and what’s an example?
reversible, non covalent binding of regulatory compounds. binding of the substrate causes conformational changes that affect the subsequent activity of other sites on the protein. results in a sigmoidal binding curve similar to hemoglobin.
ex: ATCase is regulated by the binding of CTP and ATP. CTP is one of the end products of the pathway ATCase in involved in and so when there is excess CTP it binds to the enzyme and negatively regulates it. When ATP binds it indicates that the cell needs more CTP and so positively regulates the enzyme.
what are some of the reversible covalent modifications made to regulate enzymes?
Phosphorylation, adenylation, acetylation, myristoylation, methylation
what are zymogens?
a inactive precursor that is cleaved to form the active enzyme. chymotrypsin is made from the inactive chymotrypsinogen by a nonreversible cleavage at 13-16 and 146-157 and 148-149. trypsin is made from trypsinogen from a similar process.
what are enzymes?
catalysts. they increase reaction rates without being used up. more are globular proteins, some are RNA
Why are biocatalysts better than inorganic catalysts?
greater reaction specificity to avoid side products
milder reaction conditions good for cells
higher reaction rates, biologically useful timeframe
capacity for regulation and control of biological pathway
apoenzyme
nonfunctional enzyme lacking a cofactor
holoenzyme
functional enzyme cofactor complex
prosthetic group
non protein complex that tightly binds to enzyme. cannot be removed without denaturing
coenzyme
complex organic or inorganic molecule that loosely bonds to enzyme and can be removed
cofactor
an inorganic ion usually. can be organic compound
what are the rues of enzymatic catalysis?
enzymes do not affect equilibrium ∆G
slow reactions face significant activation barriers (∆Gtransition). enzymes increase reaction rates (k) by decreasing ∆Gtransition.
How is ∆Gtransition lowered covalently and non covalently?
covalent: provide alternate chemical pathways for a reaction mechanism to occur
non-covalent: leverage entropy and enthalpy to reduct ∆Gtransition. enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex
What do enzymes bind best too?
the transition state. They have the strongest interactions with the transition state rather than the ground state substrate or product
what are the types of catalytic mechanisms?
acid-base catalysis: give and take protons
covalent catalysis: change ruction paths
metal ion catalysis: use redox cofactors, pKa shifters
electrostatic catalysis: preferential interactions with TS
what’s the deal with acid base catalysis?
Specific acid-base catalysis: uses only the H+ or OH- ions present in water
General acid-base catalysis: uses weak acids and bases other than water.
In both cases, a reaction intermediate is formed that has an unstable charge and will therefore rapidly break down to reactants. The acid-base catalysis works by transferring a proton to or from the unstable intermediate to stabilize it.
what’s the deal with covalent catalysis?
a transient covalent bond is formed between the enzyme and the substrate. This is the only catalysis that changes the reaction pathway.
requires a nucleophile on the enzyme (serine, thiolate, amine, or carboxylate
what’s the deal with metal ion catalysis?
involves a metal ion bound to the enzyme. the ion stabilizes negative charges and participates in oxidation reactions
describe the mechanism of chymotrypsin
substrate binds and nestles in hydrophobic pocket to position peptide bond for attack
interaction of Ser and His form nucleophilic alkoxide on Ser. Ser ion attacks peptide carbonyl group (covalent catalysis) and creates negative charge on carbonyl O of substrate which is stabilized by H bonding (from Ser and Gly) in oxyanion hole.
The intermediate collapses with reformation of C O double bond and breaking of the peptide bond (facilitated by His donating a proton in general acid catalysis)
A water molecule is deprotonated by general base catalysis to generate nucleophile. Nucleophilic attack forms oxyanion on substrate again, stabilized by H bonds of Ser and Gly.
collapse of this intermediate reforms C O double bond (forming product) and removes Ser bond. Ser is protonated by His to reform OH group.
Second product dissociates and enzyme is regenerated.
six classes of enzymes
oxidoreductases: transfer electrons
transferases: group transfer reactions
hydrolases: hydrolysis reaction (functional group to H2O)
lyases: cleavage of C-C, C-O, C-N by elimination
isomerases: transfer of groups within molecules to yield isomeric forms
ligases: formation of C-C, C-O, C-N by condensation coupled to ATP/cofactor cleavage
