Chapter 6 Flashcards
what were biochemical rxns originally believed to be
inseparable from life
what is vitalism
its the belief that living things are the fundamentally different from non-living things; that they contain some non-physical element and are governed by different principles that inanimate objects
what did eduard buchner demonstrate
that dead yeast still convert sugars into alcohol, indicating the reactions of life were separate from life
where is the word enzyme from
from greek it means “in yeast”
although the protein factor of some enzymes is fully active, other enzymes require…
cofactors or co enzymes for activity
what are co factors
in organic ions (Mg 2+, Fe2+, etc…)
what are co enzymes
complex organic molecules (vitamins)
what is a prosthetic group
a co enzyme or co factor that is tightly associated with the enzyme
different enzymes that use the same enzymes…
usually preform similar types of reactions
apoenzyme + cofactor/co enzyme =
holoenzyme (biologically active from)
why do we need to take vitamans
because its a molecule required by the body that cant be produced by the body
what do catalysts do
-lower the amount of energy required for a reaction to proceed
-speed up attainment of equilibrium, but do not change equilibrium
-are unchanged by the reaction; recycled to participate in another reaction
enzymes dont make a impossible reaction possible they…
take a already possible reaction and make it faster
how does catalyst compair to enzymes interms of speed
enzymes are often much faster than chemical catalysts, some approaching catalytic perfection
how does catalyst compair to enzymes interms of conditions
many chemical catalysts that require extremes of temperature, pressure and pH while enzymes function under physiological conditions
how does catalyst compare to enzymes interms of specificity
enzymes have a higher degree of specificity (incuding steriospecificity) than most chemical catalysts. this includes specificity for what they act apon and what they produce
how does catalyst compare to enzymes interms of regulation
unlike chemical catalyst many enzymes are responsiveness to the dynamic needs of the cell and organism
what is a anabolic reaction
building stuff up
what is a catabolic reaction
breaking stuff down
what enzymes are more spicific? ones that preform anabolic or catabolic reactions
enzymes preforming anabolic rxns
what is a substrate, product and active site
-substrate is the molecule acted upon by the enzyme
-product is the molecule produced by the enzyme
-active site is the portion of enzyme responsible for binding the substrate to formation of an enzyme-substrate complex
enzymes convert substrates into products but also…
convert products into substrates
what is the active site of an enzyme
3D cleft formed from the different parts of the polypeptide chain
how much of the enzyme does the active site represent
just a small portion
active sites are unique…
microenvironments
how are substrates bound to enzymes
by multiple weak interactions
what does the specificity of substrate binding depend on
the precisely defined arrangement of atoms in the active site
can enzymes and their active sited be flexible
yes
what can substrate binding cause
induced fit or conformational selection
why is hand in glove analogy better then lock in key analogy for enzyme specificity
because when you put ur hand in glove it canges the conformation and it is a perfect fit
when is a reaction spontanious
only when the delta G is negative
what is a spontaneous reaction
when a reaction will proceed with out the input of energy and the reaction releases energy (exergonic)
what if a reaction cannot take place spontaneously
then the delta G is positive and in imput of free energy is required to drive such reactions (endergonic)
what is the delta G at equilibrium
in a system at equilibrium there is no net charge in the concentrations of the products and reactants and the delta G is at 0
what does the delta G of a reaction dependent on
only on the free energy of the product minus the free energy of the reactants. The delta G of a reaction is independedt of the steps of the transformation
how come the delta G doesnt tell us anything on the rate of the reaction
because a negative delta G indicates that a reaction can take place spontaneously but does not signify weather or not it will proceed at a perceptible rate
what does the activation energy between substrate and product determine
the rate at which equilibrium is reached
what do enzymes do between substrate and product
they provide a lower-energy pathway between the substrate and the product, lowering the activation energy
what is the relationship between the rate of a reaction and activation energy
it is inverse and exponential
what determines the equilibrium of the reaction
the difference between S and P
do enzymes influence the difference in free energy
no, there for they dont influence the equlibrium
what two ways can enzyme forces lower activation energy
catalytic capabilities of enzymes results from both
-chemical effects (1 acid/base catalyst, 2 covalent catalyst)
-binding effects (1 substrate binding, 2 transition state stabilization)
what does the binding of substrate in the active site provide
specificity and catalytic power
how much can catalytic mechanisms limited to binding properties increase reaction rates
by over 10,000-fold
is there a overlap between substrate binding and transition state binding
yes there is a conceptual overlap
what do we need for an enzyme in terms of specificity
-must be complementary enough for the substrate to ensure specificity (meaning one substrate and one substrate only)
-at the same time it needs to be different enough from the substrate to promote change within the substrate
how do substrates binding promote reactions
by
-reducing entropy (decreased freedom of motion of two molecules in solution)
-alignment of reactive functional groups of the enzyme with the substrate
-desolvation of the substrate (demoval of water molecules) to expose reactive groups
-distortion of substrates
-induced fit of the enzyme in response to substrate binding
where is there an increased interaction of the enzyme and substraight
in the transition state
essence of a catalyst is stabilization of…
the transition state
how is the active site complementary to the transition state
in shape and chemical character
how much tighter may a enzyme bind to their transition states then their substraights
10^10-10^15 times more tightly
what are transition state analogs
they are stable compounds that resemble unstable transition states
are transition states of enzymes stable
no
what do transition state analogs have the potential for
therapeutic applications as competitive inhibitors
what are competitive inhibitors
they are molecules that bind to the active site of an enzyme, they tend to resemble the substrate molecule
how can transition state analogs prevent substrate binding
because they can bind the active site of a target enzyme site with high affinity
why is it better to design drug molecules that represent the transition state
-because itll bind to enzyme with much higher affinity
-youll have to use less drug molecule into the pacient
-the less you put in the lower probability itll react with something else causing some type of side effect
-cheaper
what are some ways that pathogens are interacting with hosts
in very complex ways for example:
rabies, spread from saliva to blood, host wants to spread to next victim, would do this by causing the victim to want to bite
what kind of side chains does the active site often contain
chemically reactive side chains such as Asp, Glu, His, Cys, Tyr, Lys, Arg, and Ser
what are two commonly observed mechanisms of chemical catalyst
acid/base catalyst
covalent catalyst
what is acid base catalysis reaction acceleration achieved by
catalytic transfer of a proton
in acid base catalyst the side chains can act as either… or…
bases (acceptors) or acids (donors)
histidine is often involved with…
with a pKa near physiological pH is often involved in acid/base catalysis
what is the pKa of a functional group influenced by
chemical microenvironment
what makes functional groups more suitable for acid base catalyst
functional groups from amino acids can have different pKas within the active site which makes them more suitable for acid/base reactions
covalent catalyst often involves two steps what are they
the first which forms a covalent linkage to the enzyme, the second to regenerate the free enzyme
what is kinetics
the study of the velocity of reactions
how is the velocity of a reaction quantified as
the change in concentration of product overtime V=delta [P]/delta t
what units in enzyme kinetics measured in
inits of concentration over time, for example mmoles/sec or moles/min
as enzymes are proteins any variable that influences protein structure…
my influence enzymatic activity
what is the activity of enzymes sensitive to
pH and tempature
what is enzyme velocity influenced by
enzyme and substrate concentration
for kinetics what relationship are we most interested in
the relationship between velocity and substrate concentration
when would you need induction of expression
at times of increases biological activity
what happens if there is an access of enzymes around
they can be targeted for distruction
velocity is defined as change in product concentration over time. when is it necessary to measure product
it is necessary to measure product formation before equilibrium is reached
what is initial velocity (Vo)
the velocity at the beginning of an enzyme catalyzed reaction, prior to product accumulation
what does K1 and K-1 represent
rapid, non-covalent interactions between enzyme and substrate
what is K2
its rate constant of formation of product from ES
why can you measure kinetics at equilibrium
because there is no change in conc of products overtime
how do you calculate initial velocity (Vo)
Vo=[ES]K2
how do you calculate the velocity of a reaction
V=delta[R]/delta t=[ES]K2
what happens when E+S interact (for a reaction)
E+S (K1–> or <–K-1)ES (–>K2) E+P
when a system is trying to reach eq what kind of reactions are happening
it is all about converting substrate into product with very little back reaction (<–)
what is the steady assumption
E+S (K1–> or <–K-1)ES (K2–> or <–K-2) E+P
what does the steady state assumption mathematically state
[E][S]K1=[ES]k-1+[ES]k2
in deriving the michaelis and menton equation what assumption did they work from
that the rate of formation of the ES complex was equal to the rate of its break down
what is the rate of formation of the ES complex
[E][S]k1
what is the rate of break down of the ES complex
[ES]k-1 + [ES]k2
what does the michaelis-menten equation describe
the relationship between substrate concentration and initial velocity
what is the Michaelis-menten equation
Vo=Vmax[S]/Km+[S]
the more substrate you present to a enzyme…
the faster it can use to product but it levels off
what is the Km on a Michaelis-menten plot
it is the concentration of substrate required to reach 1/2 Vmax
what is Kmax on a Michaelis-menten plot
it is the maximun velocity of the enzyme
when does velocity become independent of substrate concentration
at Vmax
the lower the km…
the higher the affinity of that particular enzyme for that particular substrate
what is 1/2 Vmax not…
1/2 the Vmax
not a measure of velocity
what is 1/2 Vmax a measure of
substrate concentration
what does the ES complex depend on
the conc of the enzyme, substrate and the K1 rate constant
for many enzymes Km provides an accurate approximation of…
the in vivo substrate concentration
if the Km provides a accurate approximation of the in vivo substrate what does this say
that most enzymes are usually functioning at about half there max velocity
when [S]<Km…
enzymes are highly sensitive to changes in substrate concentration but have very little activity
when [S]>Km…
enzymes have high activity but are insensitive to changes in substrate concentration
when [S]=Km…
enzymes has significant activity and is responsive to changes in substrate concentration
michaelis-menten sample question 1: what is the velocity of a reaction when substrate concentration is = to Km
step 1: pick values of Km and [S] that are consistent with the question here we can say Km and [S] are = to 2 mM
step 2: substitute these vales into the equation Vo=VmaxX(2mM)/2mM+2mM
step 3: express the velocity as a fraction of Vmax
Vo=2Vmax/4 Vo=1Vmax/2
michaelis-menten sample question 2: what is the velocity of a reaction when substrate concentration is double Km
step 1: pick values of Km and [S] that are consistent with the question here we can say Km is = to 2 mM and [S] is = to 4 mM
step 2: substitute these vales into the equation Vo=Vmax X 4mM / 2mM + 4mM
step 3: express the velocity as a fraction of Vmax
Vo=4Vmax/6 Vo=2Vmax/3
michaelis-menten sample question 3: what is the velocity of a reaction when substrate concentration is 1/3 of Km
step 1: pick values of Km and [S] that are consistent with the question here we can say Km 3mM and [S] is = to 1 mM
step 2: substitute these vales into the equation Vo=Vmax X (1mM) / 3mM + 1mM
step 3: express the velocity as a fraction of Vmax
Vo=1Vmax/4
how does lineweaver-burke compare to the michaelis-manten
lineweaver-burk plots are more precise method of analysis of kenetic data
what is a lineweaver-burke
-describes the relationship between [S] and V0
-double-reciprocal plot of 1/Vo vs 1/[S]
-used to determine Vmax and Km
what is the enzyme turn over number
-also called Kcat
-equals the number of molecules of substrate converted to product per unit time under saturating conditions
how do you calculate the enzyme turnover number
Vmax / [Et]
what is a enzyme inhibitor
a compound that binds to an enzyme to interfere with activity
what do enzyme inhibitors prevent
the formation of ES or the breakdown to E and P
how do reversible inhibitors bind to the enzyme
by non covalent interactions
what are the classes of reversible enzyme inhibitors with different mechanisms and kinetic consequences
-competitive
-uncompetitive
-noncompetitive
what do competitive inhibitors resemble and how do they function
the substrate and compete with the substrate for binding of the active site
competitive inhibitors only bind…
free enzymes
how can the effects of competitive inhibitors be over come
with an access of substrate (washing out)
how does a competitive inhibitor compare to substrate with no inhibitor present
Vmax is the same but apparent Km is increased
uncompetitive inhibitors bind only to…
the ES complex
how is Vmax decrease for uncompetitive inhibitors
by the conversion of ES to ESI which cannot form product
what do uncompetitive inhibitors reduce
[ES]
in uncompetitive inhibitors what happens as E binds to S to replenish ES
this apparent increase in affinity of the E for S causes a decrease in Km
what is round up
a uncompetitive inhibitor of a plant enzyme involved in amino acid metabolism
increased affinity of an enzyme would…. the Km
Shift a decrease in Km
what do uncompetitive inhibitors do to Vmax
decrease Vmax of an enzyme (so passes through graph at a higher point)
what do non competitive inhibitors bind to
E and ES (bind a site distinct from active site)
how do noncompetitive inhibitors effect Vmax and Km
Vmax is decrease with no change in Km
non competitive inhibitors dont influence S binding therefor…
there is no change in Km
what do non competitive inhibitors essentially do
essentially they reduce the number if active enzyme molecules
what does serine proteases serve as
it serves as digestive enzymes,
including trypsin, chymotrypsin, and elastase, that cleave peptide bonds in protein substrated
what does the members of the serine protease family share
simular sequences of active site residues
where are members of protease family synthesized and stored
they are synthesized and stored in the pancreas as inactive zymogens to prevent damage to cellular proteins
when and how are inactive zymogens activated
at the appropriate time by selective proteolysis
what kind of catalytic mechanism occurs with serine proteases
catalytic mechanism contains elements of both covalent and acid-base catalysis
serine proteases have unique specificities that reflect…
unique substrate binding pockets
what does thrombin cleave
Arg-Gly bonds
what does trypsin cleave by
Lys and Arg
what does Chymotrypsin cleave by
Phe, Tyr or Met
what does Elastase cleave by
Gly and Ala
what does papain cleave
it cuts all peptide bonds
what does serine proteases have a conserved catalytic mechanism based on
a catalytic triad of residues (Asp, His, Ser)
what does His act to do in serine proteases catalytic mechanism
acts to accept and donate a proton at each of the two stages of the reaction mechanism (acid base catalyst)
what does Asp act to do in serine proteases catalytic mechanism
it stabilizes the positively-charges His to facilitate serine ionization
what does Ser act to do in serine proteases catalytic mechanism
it attacks the carbonyl group of the peptide bond to be cleaved (covalent catalysis)
out of the serine protases catalytic triad of residues (Asp, His, Ser) which one is the least important
Asp
what is phase 1 of the chymotrypsin mechanism
step 1: (acid/base) His acts as base to extract proton from hydroxyl of Ser. This activates the oxygen of the hydroxyl group
step 2: (covalent) formation of a covalent linkagefrom the hydroxyl group of the Ser to the carbonyl carbon of the peptide bond to be cleaved in the substrate
step 3: (acid/base) His acts as an acid to donate a proton to the amine group of peptide bond to be cleaved, this cuts the substrate peptide into two pieces
what is phase 2 of the chymotrypsin mechanism
step 1: (acid/base) His acts as a bas to extract a proton from a water molecule activating the oxygen of this molecule
step 2: (covalent) activated water molecule attacks the point of covalent linkage between enzyme and substrate
step 3: (acid/base) His acts as an acid to donate a proton to reform the hydroxyl group of Ser
what are some ways you can tell the different phases of the chymotrypsin mechanism
-if polypep chain is still in one piece its phase 1 if in two phases its in phase 2
-differs in where His gets/donates its protons. in phase 1 it gets a proton from ser in phase 2 it gets a proton from h2o molec
-phase 1 it donates it onto the peptide bond phase 2 it donates it onto the peptide bond phase 2 it donates it onto the hydroxyl group of ser
how can the activity of an enzyme be regulated by
controlling the amount of the enzyme (long term) or by the activity of a constant quality of the enzyme (short term)
what can regulate the enzyme availability
location, rates of synthesis and degridation
what can regulate the enzyme activity
a) covalent modifacation
-phosphorolation, methylation, glyosylation etc
b) non-covalent modification (allosteric)
-allosteric regulation
enzymatic reactions are often controlled through _______ by the final product of the pathway
negative feedback inhibition
what often inhibits the enzyme and what step is it
the final product often inhibits the enzyme catalyzing the first unique and committed step
in regulation of enzymatic activity what does inhibiting the the first unique and committed step prevent
regulation at this step conserves material and energy and prevents accumulation of intermediates
in points of regulation describe the roles of steps A(e1)B(e2)C(e3)D(e4)E(e5)F
-F is the end product it is needed in limited amounts and cannot be stores
-A is valuable and showed to be conserved unless F is needed
-BCD and E have no biological roles other then as an intermediate in production of F
how does the negative feed back in a branched pathway often occur
by the final product of each branch acting to inhibit the enzyme catalyzing the first unique and committed step of the branch
what happens in regulation when two pathways cooperate to form a single product
-the final product can inhibit the first unique step of each branch
-the molecules preceding the merger can inhibit the first step if their branch as well as activating the first step if the opposing branch
what do allostaric enzymes do
they serve as information sensors to coordinate cellular metabolism
how are allosteric regulated
-interactions with metabolic intermediates
-allosteric modulators that bind non-covalently at sites other then the active site
what structures are allosteric usally
usually examples of quantinary structures
what kind of reactions do allosteric enzymes catalyze
branch point reactions
because allosteric enzymes reactions are slow this represents…
rate limiting steps of the pathway
allosteric enzymes do not obey _____, instead they have ______ curves
michaelis-menten kinetics, sigmoidal curves
allosteric enzymes transition from a…….within a ……….
less active state to a more active state, narrow range if substrate concentration
the activity of allosteric enzymes is more sensitive to changes in….
substraite concentration near the Km than are the michaelis-manten enzymes of the same Vmax
decribe the sensitivity in allosteric enzymes called threshold effect
below a certain substrate concentration there is little enzyme activity; after the threshold has been reached the enzyme activity increases rapidly (on/off)
what does PFK1 catalyze (phosphofructokinase)
an early step of glycolysis
what is an inhibitor and activator of phosphofructokinase
phosphoenolpyruvate (PEP) is an intermediate near the end of the pathway. it is an allosteric inhibitor of PFK1
ADP is an allosteric activator of PFK1
what happens when ratio [PEP]/[ADP] is high
PFK1 is inhibited
what happens when [PEP]/[ADP] is low
PFK1 is activated and glycolysis produces more ATP from ADP
alostaric inhibitors are rate….
limiting steps
the activity of PFK1 is responsive yo the concentration of…
substrate as well as the allosteric activators and limitors
even at levels of substrate the activity of the enzyme can be through changes…
in levels of the allosteric modulators
many enzymes are regulated through…
covalent linkages of a modifying group to change some aspect of the proteins behavior such as activity
the most common post translation modifacation is…
through phosphoralation
is enzyme regulation by covalent modifacation reversable
yes they are usually reversable with one enzyme catalyzing the addition of the group and another enzyme catalyzing the its removal
what adds phosphoryl groups and what removes them
kinase adds phosphoryl and phosphates removes them
what two enzymes are responsible for the production and utilization of glycogen
-glycogen synthase (anabolic) which catalyzes production of glycogen from glucose
glycogen phosphorylase (catabolic) which catalyzes the breakdown if glycogen into glucose
what is it called if you had both anabolic and catabolic reactions occurring at the same time
its called futile cycling
how do you avoid futile cycling
through reciprocal regulation ensuring that only anabolic or catabolic is happening at any given time
what happens to glycogen phosphorylase and glycogen synthase in response to hormones released when your scared or hungry
both enzymes are phosphoralated. which activates the catabolic enzyme and inactivates the anabolic enzymes
in glycogen metabolism what reaction if favored when in the fed state and un fed/scared state
unfed/scared: glycogen into glucose
fed state: storage og glucose within glycogen
what happens in response to glycogen phosphorylase and glycogen synthase when hormones released when the body has been fed in glycogen metabolism
both enzymes are unphosphorylated
in glycogen metabolism when unphosphoralated the anabolic enzyme is____and the catabolic enzyme is____
active, unactive
