Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
168 Cards in this Set
- Front
- Back
|
coenzymes
|
derived from vitamins
vitamin deficiencies are due to the loss of one or more enzyme activitis that req a particular enzyme broken into activation-transfer coenzymes and oxidatrion-reduction coenzymes |
|
|
2 groups of coenzymes
|
1. activation-transfer coenzyme
a. thiamine pyrophosphate coenzyme A biotin pyridoxal phosphate 2. the oxidation reduction coenzymes FAD NAD+ Vit C Vit E |
|
|
thiamine pyrophosphate
|
action-transfer coenzymes
syntehsized in hyman cells by adding pyrophosphate onto thiamine fnal group of TPP is a reactive C atom that readily forms carbanion it forms a covalent bond with a substrate keto group resulting in the cleavage of the neighbring C-C bond |
|
|
coenzyme a
|
CoASH is syntehsized from vit pantothenic acid (vit B5)
phosphopantothenic acid is linked to adenosine 3'5' biphosphate (parts of CoASH) CoASH binds reversibly to an enzyme active site posses a sulfydryl group that participates in acyl transfer rxn through the formation of highly reactive thioesters acytl COA plays an important role in Citric acid cycle by donating 2 C units that can be oxidized to CO2 |
|
|
thiamine deficiency
|
observed in alcoholics - vit deficiency smptoms manifested by loss of enzyme activity
limits the oxidation of alpha keto acids (TCA) leading to pleiotropic effects on nervous, cardiovascular and other organ systems |
|
|
biotin
|
active-transfer coenzyme
vitamin in its own right and req in human diet attached to enzymes through epsilion amino group of an active site Lys residue (ex pyruvate carboxylase |
|
|
pyridoxal phosphate
|
active-transfer coenzymes
PLP syntehsized from pyridoxine (Vit B) - contains a reactive aldehyde that readily forms a schiff base w/ primary amine -present on enzyme or on substrate molecule PLP dependent enzymes are involved in transamination, racemization, and decarboxylation rxns glutamate amino transferase which interconverts L-glutamate and alpha ketoglutarate is ex of a PLP dependent enzyme |
|
|
NAD+
|
syntesized from the vitamin Niacin
NAD+ dependent dehyydrogenases catalyze the transfer of hydride ion from C to C4 position of nicotinamide ring the positively charged pyridine ring is a stronger electrophile permitting the transfer of the hydride ion lactate dehydrogenase an NAD dependent enzyme interconverts lactate and pyruvate NAD+/NADP+ has a role in 2 electrn transfers |
|
|
liver alcohol dehydrogenase
|
NAD+ dependent enzyme
catalyzes the oxidation of ethanol to acetaladehyde in liver active site Ser removes a proton from ethanol to form an anion that is stabilized by enzyme bound zinc atom which leads to transfer of a hydride ion to NAD+ to form NADH 90% of acetaldehyde produce by alcohol dehydrogenase is further oxidized to acetate by acetaldehyde dehydrogenase (ALDH), another NAD+ dependent enzyme this acetate can then enter the Citric Acid Cycle |
|
|
enzyme kinetics
|
central approach to understanding the mech of enzyme catalysis
involves det the rate of a rxn under a variety of exp. conditions (pH, substrate conc, or changed residues of an active site conc of sybstrate is key factor affecting the rate of the rxn lower [S], vo (initial velocity) increase almost linearly w/increases in the concentration of S at higher [S] the increase in vo becomes smaller and smaller until a point is reached where further increase in [S[cause only sml increase in vo Vmax = maximum velocity an asymptote to this plateau Vmax occures when [S]>>[Et] at Vmax all of the enzyme active sites are occupied w/either substrate or product when this occurs enzyme is saturated and reaction rate only depends on [Et] (doubling this would double the rate of rxn) each step of a rxn has it's own rate constant and the overall rate is proportional to the concentration of ES and at steady state the concentration of ES complex is constant this forms the basis of the steady state assumption - the rate of ES formation is equal to the rate of ES breakdown |
|
|
michaelis menten eqn
|
vo is the initial rate during the steady state - steady state kinetics
plots of initial velocity versus [s] typically adopt the form of a rectangular hyperbola that is descrived by the michaelis menten eqn: vo= Vmax[s]/Km + [S] Km is concentration of substrate where the vo is equal to 1/2 Vmax - is equal to (k-1 +k2 / k1) - a dissociation constant for the interaction of the substrate w/the enzyme - not true b/c contains the k2 term - so smlr the Km the higher the substrate binding affinity |
|
|
turnover number
|
kcat = Vmax/[Et]
[Et] = total enzyme concentration units are s^-1 equivalent to the number of substrate molecules turned over to product in a given unit of time by a single enzyme molecule at sturating conc of substrate tells you how the enzyme responds to the substrate under saturated conditions - how efficient |
|
|
specificity constant
|
Kcat/Km
used to compare enzymes 2nd order rate rxn when [S]<<Km the michaelis menten eqn reduces to: vo=(kcat/Km) [Et][S] under these cond. vo is dependent on [Et] and [S] kcat/Km is usually expressed in units of M-1s-1 the upper limit to the value of kcat/Km is 10^9M-1s-1 limit due to the fact that 2 molecules cannot diffuse together faster than the rate of diffusion |
|
|
lineweaver-burk
|
aka double reciprocal plot
used to graphically determine valuse of Km and kcat the double reciprocal of the michaelis menton eqn discribes the line on this graph too vo= Vmax[S]/[S]+Km which gives the lineweaver burk eqn 1/vo= Km/Vmax[S] +1/Vmax x-axis =1/[S] y-axis = 1/vo slope = Km/Vmax 1/[S] axis intercept = - 1/Km 1/vo axis intercept = 1/Vmax allows more accurate determination of kinetic constants than vo vs [S] |
|
|
hexokinase
|
an ex of an isozyme
hexokinase exists as a number of tissue specific isozymes a graph can compare their velocity of different isozymes (hexokinase I RBC and hexokinase IV (glucokinase from liver) the graph has glucose conc on x-axis and vo/Vmax as y axis the glucose Km of hexokinase I and other isozymes found in skeletal mm and brain in the range of 0.02 to 0.13 mM glucokinase has a much higher Km for glucose (~5mM) and - sigmoidal curve using this info the concentration of S that yields half max activity (S0.5) is similar at 6.7mM around liver cell cange in glucose concentration around Km changes the velocity but it doens't really chagne in RBC blood glucose - 5mM this means enzyme (in liver) responds quickly to change in glucose concentration if concentration drops enzyme activity drops so it will keep the liver enzyme from competing w/other organs for the limiting amts of glucose and vice versa Hexokinase II is predominant in myocytes and Km for glucose is 0.1 (blood is 5) meaning that it operates at Vmax - intercellular conversion of glucose to glucose-6-phosphate in liver permits greater amts of glucose to enter the cell so that it can be used for E or stored as glycogen |
|
|
isozymes
|
have different AA but catalyze the same rxn
differences btw them reflect the role, tissue localization, cellular localization of the enzyme fetal isozymes are often different than the adult form of the enzyme - this form is expressed in cancerous cells differences can be exploited clinically ex- creatine kinase = electrophoresis/amt quantified - MI? |
|
|
creatine kinase
|
has 3 isozymes
CK-1 : BB found in brain CK-2: MB found only in heart CK-3: MM found in skeletal and heart mm CK catalyzes the reversible phosphorylation of creatine - phosphocreatine is an imp source of chem E in heart, brain, and skeletal m - a cytosolic enzyme that is released into blood in response to tis. damage CK-2 and CK-3 can be seberated by electrophoresis and their amts quantified increased levels of CK-2 is indicative of an MI |
|
|
Multisubstrate rxns
|
most enzymes have multiple substrates - 2 or more
ex- hexokinase catalyzes a rxn in which there are 2 substrates -ATP and glucose 2 substrate rxns analyzed by Michaelis-Menton eqn but the sequence of substrate binding and product release affects the rate of the rxn Km MAY depend on the conc of the second substrate 2 substrate rxns follow one of 2 kinetic pathways - sequential or ping-pong |
|
|
sequential kinetic pathway of multisubstrate rxn
|
- occur when both substrates are bound to theenzyme at the same time - ternary complex formed
apparent Km decreases as the concentration of the seconds substrate increases until an infinite conc of S2 is reached [S2] is held constant and [S1] is varied done at different fixed levels of S2 to get different graphs Vmax - increases -larger [S2] lower y-axis intercept on a lineweaver burk plot Km decreases you can regulate the activity of this kind of enzyme by varying the conc of any one if its substrates the x axis intercept is further to the right as substrate increases |
|
|
ping pong kinetic pathway of multisubstrate rxn
|
involve 2 substrates
1st contains a reactive group that is first transfered to an active site residue that substrate leaves and the second substrate binds and the fna; group on the enzyme is then transfered to the second substrate many enzymes use acetyl-coenzyme A as a substrate in this type of rxn parallel lines on the lineweaver burk plot are indicative apparent Km inc as conc as the 2nd substrate inc until an infinite conc of B is reached As B inc y-intercept lower and x-intercept more to the left |
|
|
enzyme inhibition
|
enzymes are subject to both reversible and irreversible inhibition
inhibitors are cmpds that interfere w/enzymatic rxn such that the velocity of the rxn is either decreased or completely abolished reversible bind noncovalently and irreversible covalently modify the enzyme |
|
|
reversible inhibiton
|
3 general types
1. competitive 2. noncompetitive (mixed) 3. uncompetitive |
|
|
Competitive inhibition
|
an ex- of reversible inhibitor
compete w/substrate for binding to the active site structurally similar to substrate combine w/enzyme to form the enzyme-inhibitor complex wich dec. conc of free enzyme dec the rate of rxn and blocks the substrate from binding rxn rate can be inc in the presence of the inhibitor by inc the amt of substrate therby reducing the prob the inhibitor will bind to enzyme at active site |
|
|
competitive inhibition lineweaver = burk eqn
|
when [S]>>[I] rxn has norm Vmax
michaelis-menton eqn => vo= (Vmax[S])/(a*Km+[S]a0 where a = 1+ ([I]/Kis lineweaver burk - 1/vo=aKm/Vmax[S]+1/Vmax when no competitive inhibhitor alpha=1 lines intersect at 1/Vmax b/c inc the subs conc means S will eventually outcompete I for binding A competitive inhibitro doesn't change Vmax however as I conc inc the apparent Km inc Smlr 1/Km value) due to changes in slope of the line |
|
|
uncompetitive inhibition
|
ex of a reversibile inhibition
inhibitor binds to a site that is distinct from the site of binding of substrate this leads to the formation of the enzyme-substrate-inhibitor complex formation of this complex dec the conc of ES therby dec the rxn rate and Vmax |
|
|
uncompetitive inhibition lineweaver = burk eqn
|
in the presence of an uncompetitive inhibitor
vo=Kmax[S]/Km+a^1[S] a^1= 1+[I]/Kii 1/vo= Km/Vmax[S] + a^1/Vmax a parrallel pattern of lines is indicative decrease Vmax and apparent Km b/c the [S] that is req to reach 1/2 Vmax dec by a factor of (1+[I]/Kii) or a^1 |
|
|
Noncompetitive inhibitor
|
inhibitors bind to a site that is different from the substrate binding site but it can either bind to either E or ES which leads the formation of both the EI and ESI complexes
formation of both causes Vmax to dec |
|
|
noncompetitive inhibitor lineweaver-burke
|
vo= Vmax[S]/aKm+a^1[S]
1/v= a^1/Vmax + aKm/Vmax[S] an intersecting pattern of lines where the intersect lies to the left of the y-axis is diagnostic Vmax lower in the presence of inhibitor if the inhibitor has equal affinity for E and ES (Kis=Kii) then the lines will intersect at the x=axis no affect on Km typically act on multisubstrate enzyme the kinetics can be much more complex than abv depending on whether I binds at one of the substratebinding sites if so it mayb be in competitive w/the substrate but noncompetitive w/the other - in these cases Km could vary |
|
|
reversible inhibition summary
|
competitive inc Km
uncompetitive dec Km and Vmax noncompetitive dec Vmax |
|
|
mechanism based inhibitors
|
act non-specifically or very specifically and often lead to the covalent modicfication and inactivation of the enzyme
mech based inhibitors - 2 classes 1.covalent 2. suicide substrates(inhibitors |
|
|
covalent inhibitors
|
best and only way to "reverse' the effects of a covalent inhibitor is to synthesize more enzyme
ex- organophosphorous cmpds react w/ active site Ser residues in enzyme such as the ser proteases and acetylcholinesterase penicillin is one too |
|
|
penicillin
|
ex of covalent inhibitors along with other beta-lactam containing antibiotics
-posses a beta-lactam ring which posses a thiazolidine ring attached to it - the structures differ at the 6th position penicillin inhibits an enzyme called transpeptidase that catalyzes the terminal step in bacterial peptidoglycan synthesis - an essential protein-polysaccharide component of bacterial cell walls - the final step involves formation of an amide bond btw the terminal Gly residue at the end of one chain with the D-ala on another chain so the terminal D-ala residue is displaced ultimately this rxn generates a cross link btw 2 peptidoglycan chains however when penicillin binds to traspeptidase an active site Ser is deprotonated by the ring N in penicilin which leads to the generation of the nucleophilic alkoxide anion the Ser alkoxide attacks the carbonyl C leading to a stable penicilloyl-enzyme complex the complex can no longer catalyze the transpeptidation rxn beta lactamases are also covalently modified by beta lactam antibiotics but these enzymes are designed to hydrolyze the penicilloyl-enzyme intermediate - mech in which bacteria prevent the bactercidal effects of penicillin |
|
|
net yield of glycolysis
|
2 ATP
2 NADH (which can each go on to provie an additional 1.5 molecules of ATP via ETC) |
|
|
major sources of acetyl group for acetyl coA
|
Fatty acids through Beta-oxidation
ketone bodies degraded glucose through glycolysis ala and ser through AA breakdown ethanol through ethanol detox |
|
|
Citric Acid Cycle
|
opperates w/n mitochondria
accounts for over 2/3 of ATP generated during the oxidation of food major E-generating metabolic pathways (glycolysis, fatty acid oxidation, and ketone body oxidation) converge on Acetyl Co-A the acetyl group of acetyl CO-A contributes 8 e- to the CAC and 2 C atoms these 8 e- are transferred to 1 molecule of FAD and 3 molecules of NAD+ to form the reduced form of these coenzymes after several turns of the cycle the 2 C atoms from the original acetyl group are converted to 2CO2 although it looks like 2 are given off they really arent b/c C are accounted for but the O aren't - only 1 O comes from actyl CoA other 2 comes from H2O and 4th one comes from the Pi bonding with GDP acetyl CoA + 3 NAD+ + FAD -> 2CO2 + CoASH + 3 NADH + 3H+ also + GDP + Pi + 2 H2O -> + FAD (2H) + GTP |
|
|
production of acetyl CoA from Pyruvate - CAC
|
Pyruvate dehydrogenase complex (PDC) catalyzes the conversion from pyruvate to acetyl-CoA
an alpha ketoacid dehydrogenase family of enzymes the basic rxn involves the oxidative decarboxylation of pyruvate to form a high E thioester linkage irreversible PDC has 3 catalytic subunits -pyruvate, CoA-SH, NAD+ PDC is composed of 3 mitochondrial enzymes 1. Pyruvate decarboxylase (E1) (+TPP) 2. Dihydrolipoyl transacetylase (E2) (+lipoate) 3. Dihydrolipoyl dehydrogenase (E3) (+FAD) overall rxn req 5 coenzymes. 2 e- are produced during the NAD+ to NADH and are donated to the ETC where they are used to produce 2.5 molecules of ATP pyruvate is an alpha ketoacid |
|
|
Steps of CAC
|
1. The TPP carbanion acts as a nucleophile and attacks the carbonyl C of pyruvate. Decarboxylation produces a stabilized carbanion
2.this carbanion attacks an oxidized molecule of lipoate (lipoyl-lysine) attached to the E2subunit of the PDC. Ultimately an acetyl thioester of reduced lipoyllysylis formed 3.E2 catalyzes the transfer of the acetyl group to CoA to generate Acetyl-CoA 4. E3 (dihydrolipoyl dehydrogenase) catalyzes the oxidation of the lipoate group to regenerate oxidized lipoate. The e- are transfered to FAD to form FADH2 5. E3 transfers the e- from FADH2 to NAD+ to form NADH |
|
|
the PDC- lipoic acid
|
lipoate or lipoic acid is a coenzyme that is not synthesized from a vitamin precursor
covalently linked to the epsilion amino group of a lys side chain in E2 which produces long flexible apendage that can translocate great distances - can move btw active sites distance is greater than or equall 5nm the long flexible arms of coenzym A and biotin are often employed as flexible appendages that link distant active sites |
|
|
regulation of PDC
|
1.phosphorylation
2. allosteric mech PDC Kinase catalyzes phosphorylation of Ser residuces on E1 to inactivate PDC PDC kinase is inhibited by ADP and pyruvate -so when ADP levels rise due to inc utilization of ATP the kinase activity is inhibited and PDC remains active to provide Acetyl-CoA for the CAC when pyruvate levells rise signaling an abundant source of 2 C units PDC kinase is again inhibited and PDC remains active to provide acetyl CoA for the CAC Ca+ ion in heart stimulates PDC kinase( inc ) Ca+ rises during contraction |
|
|
overall yield of Citric acid cycle
|
3 NADH, 1 FADH2, 1 GTP
|
|
|
intermediates of the CAC
|
used for the synthesis of other products and E producing NADH FADH2, and GTP
use of intermediates by other pathways means the intermediates must be replenished in order to maintain fn integrity of the cycle. this is accomplished by anaplerotic(replenishing) replenishing rxn |
|
|
suicide substrates
|
inhibitors that use the suicide substrate mechanism
|
|
|
gout
|
caused by the precipitation of uric acid in the joints
urate is formed during the metabolism of purine nt (AMP+GMP) allopurinol is a sucide substrate that inhibits Xanthine oxidase(XO) and is used to treat gout XO catalyzes 2 rxns at the same active site 1st is hydroxylation of hypoxanthine to form xanthine which is then oxidized to form urate XO catalyzes the oxidation of allopurinol to alloxanthine a cmpd that remains tightly bound to the enzyme (t1/2 of the dissociation of the drug is 300 min) AMP-> hypoxanthine -XO-> Xanthine -XO->urate |
|
|
Enzyme regulation
|
flux through metabolic pathways is often controlled by one enzyme in a sequence of rxns (a regulatory enzyme) that is either activated or inhibited
these enzymes usually catalyze rate-limiting steps in pathways and exert control on the flux through the whole pathway |
|
|
mechanisms of enzyme regulation
|
1. concentrations of substrates or inhibitors
2. product concentration 3. allosteric activators and/or inhibitors 4. covalent modification 5. binding of modulator proteins 6. proteolytic cleavageb 7. enzyme level - effects on transcription or translation |
|
|
product inhibition
|
- enzyme regulation
high product concentration can dec the concentration of free enzyme and consequently dec the rate of the rxn ex- product inhibition of hexokinase by glucose-6-phosphate conserves blood glucose for tis needing it when rates of glycolysis and glycogen syn are low the conc of glucose-6-phosphate rises and inhibits the rate of the hexokinase rxn |
|
|
allosteric enzymes
|
typically contain multiple subunits and exhibit positive cooperativity
the first substrate will bind poorly to the enzyme bc all the subunits are in the low affinity T state but binding of the first substrate to the T conformation converts converts its own subunit and at least one adjacent subunit to the high affinity R state binding the second and subsequent substrate molecules converts the remaining subunits from T to R conformation in a stepwise fashion |
|
|
allosteric effectors
|
bind to allosteric enzymes
can be activators or inhibitors and bind at sites distant from the active site - often on diff subunits |
|
|
allosteric activator
|
alter the active site to inc its affinity for the substrate and inc the rxn rate
binding could cause repositioning of a side chain such that it is better able to contribute to rate enhancement or substrate binding bind more tightly to the R state - the allosteric site is only open in the R state and helps to stabilize the R confromation |
|
|
allosteric effectors
|
bind more tightly to the T state and help to stabilize that conformation
as a result the conc of either the allosteric activator or the substrate must be inc to overcome the effects of the allosteric inhibitor |
|
|
allosteric effectors kinetics
|
enzymes in the absense of effectors exhibit sigmoidal kinetics b/c binding of a substrate molecule causes a conformational change in the remaining subunits to activate them
w/allosteric activator the vo vs [S] curve tends to become more hyperbolic (shift to the left) and Km or (S0.5) tends to dec substantially b/c the activator changes all of the subunits to high affinity state doesn't change Vmax w/inhibitors the S(0.5) inc (shifting the curve to the right) required for 1/2 Vmax - making it more difficult to substrate the enzyme dec. Vmax |
|
|
K effectors
|
allosteric activators that alter Km w/o altering Vmax
|
|
|
allosteric enzymes in metabolic pathways
|
control points in key metabolic pathways
allosteric inhibitors have a greater effect on enzyme velocity than ordinary reversible inhibitors many key effectors are structurally unrelated to the substrate or products of the rxn ex - ADP activates isocitrate dehydrogenase a key enzyme of the CAC AMP is allosteric activator of glycogen phosphorylase and phosphofructokinase-1 allosteric control of both of these enzymes causes increased production of ATP either from CAC or from glycolysis allosteric control of enzyme is useful bc its effects on enzyme activity tend to be nearly instantaneous - rapid adjustment of flux |
|
|
covalent modification
|
type of enzyme regulation
ex - protein phosphorylation catalyzed by protein kinases whereas dephosphorylation is catalyzied by protein phosphatases phosphorylated on Ser, Thr, or Tyr side chains and ATP is typically the donor the bulkiness and neg charge of phosphate groups can cause conformational changes in proteins due to their ability to H-bond and participate in either attractive or repulsive electrostatic interactions w/other residues activity of protein kinases and protein phosphotases is controlled by hormone levels ex - m and liver phosphorylase - phosphorylation activates this enzyme |
|
|
glycogen phosphorylase
|
Ex - of covalent modification
catalyzes: (Glucose)n + Pi -> (glucose)n-1 + glucose-1-phosphate - a dimer of identical subunits that interconvert btw a less active and a fully active conformation depending on its phosphorylation status and teh concentration of AMP ( AMP is an indicator of cellular energy levels - when conc of AMP are high ATP levels are low phosphorylation on Ser of AMp binding will cause a conformational change in this enzyme to inc activity in m. the activity is regulated by adrenaline thus inc in adrenaline cause inc breakdown of glyocgen and thereby provide the E nec for our fight or flight response |
|
|
protein kinase A
|
PKA
-ex of covalent modification s Ser/Thr kinase that catalyzes the phosphorylation of a number of proteins in key metabolic and signal transduction pathways ex - glocogen phosphorylase kinase controled by several hormones including adrenaline and epi that act by causing the intracellular conc of cAMP to rise b/c PKA activity is regulated by the intracellular conc of cAMP it is aka cAMP-dependent protein kinase rise in intracellular cAMP the molecule binds to the regulatory subunits of PKA and cases them to disassociate which releases the catalytic domains such that the activity of the enzyme is increased in a flight or fight response, adrenaline binds to a G protein coupled receptor and indirectly causes the conc of cAMP to inc whcih inc PKA activity which results in the phosphorylation of glycogen phosphorylase kinase and consequently causes the inc in the activity of glycogen phosphorylase - ex of signal transduction cascade |
|
|
binding of modulator proteins
|
ex of enzyme regulation
regulate the activity of another protein or enzyme by binding to that protein similiar to allosteric effectors, they can exert effects by producing conformational changes or steric clashes w/substrates to either inc or dec enzyme activity ex-calmodulin, small g proteins |
|
|
calmodulin
|
active when bound to Ca2+ and then binds to glycogen phosphorylase kinase and activates it thereby stimulating the breakdown of glycogen and provding m w/fuel
during m contraction Ca2+ is released from the sarcoplasmic reticulum |
|
|
sml g proteins
|
(ex- Ras)
ex- of binding of modulator proteins for enzyme regulation regulate the activity of other enzymes through protein-protein interactions G proteins are enzymes that hydrolyze GTP to form GDP when G proteins are bound to GTP they adopt a conformation that enables it to bind to a target protein which anc activate or inhibit the activity of the interacting protein rather slow catalysits on their own and the rate at which GTP is hydrolyzed is slow but this allows them to exist in the GTP bound conformation for extended periods of time. once hydrolyzed the bound molecule of GDP is eventually replaced w/GTP activity also regulated by accessory proteins 1. GAP GTPase activating proteins - inc the rate of GTP hydrolysis - turn off signal 2. GEFs inc the rate at which GDP is exchanged for GTP ( turn on signal) 3. GDIs inhibit the dissociation of GDP turn off signal |
|
|
Ras
|
an ex of a sml G protein that plays an imp role in controlling cellular growth
a peripheral mem protein that associates w/the membrane b/c it is covalently modified by a sml lipid (a farnesyl group) the activity is regulated by SOS a GEF when SOS itself activate it promotes the exchange of a molecule of GDP for GTP once it binds to GTP it recruits and activates a Ser/Thr protein kinase named Raf to propagate the signal transduction cascade ultimately this leads to the activation of gene transcription |
|
|
proteolytic cleavage
|
ex of enzyme regulation
used to activate enzymes is proteolytic cleavage and is irreversible many are synthesized as precursor proteins or zymogens that are catalytically inactive ex-precursor forms of chymotrypsin and trypsin (chymotrpsinogen and trypsinogen) chymotrypsinogen is secreted by the pancreas and it is activated in the digestive tract by trypsin which cleaves btw Arg 15 and Ile16 allows the protein to adopt a new conformation creating binding cleft for the substrate 2nd proteolytic event in the activation is autocatalytic trypsin is activated in enteropeptidase |
|
|
enzyme level
|
regulates enzymes
since Vmax is proportional to [E] changes in enzyme level can substantially alter the rate of a metabolic rxn levels of a particular protein can be controlled by: 1. the regulation of gene transcription 2. stabilization of messenger RNA - ex inc translation 3. regulated degradation - the proteosome |
|
|
regulation of metabolic pathways
|
can be regulated by a regulatory enzyme but many pathways have intermediates or branch points where the intermediate becomes a precursor for 2 or more pathways
these can be regulated at multiple points using a variety of mech: 1. regulation of rate limiting step 2. feedback regulation 3. feed-forward regulation 4. tissue isozyme of regulatory proteins 5. counter regulation of opposing pathways 6. substrate channeling through compartmentalization enzymes can also be assembled into multi-enzyme complexes so that intermediates of the pathway are directly transfered between the active sites of 2 enzymes that are linked sequentially in metabolic pathway |
|
|
regulation of the rate limiting step
|
the slowest step of a pathway is often the 1st committed step in the pathway and is often not readily reversible
|
|
|
feedback regulation
|
the end product of a pathway may control its own synthesis by either inhibiting upstream enzyme or influencing its transcription
|
|
|
feed-forward regulation
|
the substrate of the pathway may activate the enzymes in that pathway
|
|
|
tissue isozymes of regulation
|
depending on the needs of individual cell types the cell may synthesize a tissue specific isozyme - mm and liver forms of hexokinase
|
|
|
counter regulation of opposing pathways
|
a biosynthetic pathway can have different regulatory enzymes than the opposing degradative pathway
when glycogen synthesis is activated glycogen breakdown is inhibited |
|
|
substrate channeling through compartmentalization
|
enzymes or pathways w/common fns are often assembled into organelles - the TCA cycle in mitochondrion
|
|
|
self sealing ability of membranes
|
allows them to fn in endocytosis,exocytocis, & cell division
|
|
|
composition of membranes
|
major components
1. Lipids A, phosphoacylglycerides - B. Sphingolipids I. Sphingomyelin II. glycophingolipids III. gangliosides 2. Cholesterol 3. Membrane proteins A. Integral B. Peripheral C. lipid anchored |
|
|
phosphoacylglycerides
|
basic structure is 2 fatty acids linked to a glycerol through an ester linkage
a polor head group is attached to the 3rd C of glycerol through a phosphodiester linkage a variety of diff head groups major component of biol membranes different fatty acids can be linked to the glycerol moiety therefore a particular type may exist as a number of diff molecular species inc levels of lipids containing unsaturated acyl chains in the cis conformation inc the fluidity of a membrane whereas saturated acyl chains dec the fluidity of membrane |
|
|
differing types of membranes have different compositions
|
liver large amt proteins and lesser amts of lipids (same as bacteria)
mitochondrial membranes are rich in proteins bc of a number of enzyme catalyzed process occurs across these membranes the myelin sheath is predominantly composed of lipids proteins and lipids are often attached to oligosaccharides on the external surface and may constitute 2-10% of the weight of the plasma membrane hydrophilic layer of carbs is called glycocalyx and is present to protect the cell from digestion and to control the uptake of hydrophobic cmpds |
|
|
cholesterol
|
present in most membranes but most abundant in PM and least abundant in inner mito membrane
member of the sterol family 4 fused rings role in maintaing proper fluidity of the mem it can intercalate btw packed saturated acyl chains and inc fluidity and it can interact w/unsaturated acyl chains thereby restricting their motion and decreasing fluidity |
|
|
cardiolipin
|
major component of the inner mito membrane but is absent from plasma membrane
|
|
|
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol
|
minor components of all membranes but serve specialized fns in signal transduction
|
|
|
sphingolipids
|
contains sphingosine, an amino alcohol, one lng chain faatty acid and one polar head group X
the head group is joined by a glycosidic linkage in some cases and a phosphodiester linkage in others X - could be glucose, phosphocholine, or complex oligosaccharide 3 subclasses exist : 1. sphingomyelins ex- sphingomyelin 2. glycosphingolipids ex- glucosylcerebroside 3.gangliosides ex - GM2 |
|
|
aymmetrical distribution of lipids
|
each monolayer has a different composition w/respect to individual phosphoglycerides and sphingolipids
cholesterol is equally distributed in lipid bilayer phosphatidylserine mvmt to the outer monolayer of a platelet is imp for the formation of blood clots phosphatidlserine mvmts to the outer monolayer is also imp in apoptosis affect movement and rotational ability of fatty acids |
|
|
integral membrane proteins
|
strongly associated w/the lipid bilayer and can only be extracted w/detergents, organic solvents, or denaturants
removal from bilayer often leads to denaturatino 10-20% of all proteins are these many contain single or multiple transmembrane segments so they contain one or more intracellular domains many are glycosylated on extracellular portions proteins can be glycosylated on Ser and Thr residues (O-linked glycosylation) and on Asn (N-linked glycosylation) strongly associated w/membranes b/c of hydrophobic interactions btw membrane spanning domains of the protein and the acyl fatty acid chains of the lipids a variety of topologies, tertiary and quaternary structures are utilized by integral membrane proteins to span the bilayer - i.e. multiple amohiphilic helices or beta sheet like structures |
|
|
regulation of metabolic pathways
|
can be regulated by a regulatory enzyme but many pathways have intermediates or branch points where the intermediate becomes a precursor for 2 or more pathways
these can be regulated at multiple points using a variety of mech: 1. regulation of rate limiting step 2. feedback regulation 3. feed-forward regulation 4. tissue isozyme of regulatory proteins 5. counter regulation of opposing pathways 6. substrate channeling through compartmentalization enzymes can also be assembled into multi-enzyme complexes so that intermediates of the pathway are directly transfered between the active sites of 2 enzymes that are linked sequentially in metabolic pathway |
|
|
regulation of the rate limiting step
|
the slowest step of a pathway is often the 1st committed step in the pathway and is often not readily reversible
|
|
|
feedback regulation
|
the end product of a pathway may control its own synthesis by either inhibiting upstream enzyme or influencing its transcription
|
|
|
feed-forward regulation
|
the substrate of the pathway may activate the enzymes in that pathway
|
|
|
tissue isozymes of regulation
|
depending on the needs of individual cell types the cell may synthesize a tissue specific isozyme - mm and liver forms of hexokinase
|
|
|
glycophorin
|
3 domains
1) A hydrophilic N-terminal domain that protrudes outside of the cell and contains a number of O- and N-linked sites of glycosylation (red). 2) A single 20 residue helical-like transmembrane domain. 3) A hydrophilic C-terminal domain that protrudes from the inner face of the membrane and interacts with peripheral proteins (e.g protein 4.1) that connect the protein to the membrane cytoskeleton, i.e. actin and spectrin (see previous slide). makes RBC sticky |
|
|
hydropathy plot
|
Note that a 20 residue α-helix is long enough to traverse a membrane bilayer (~ 30 Å).
Individual amino acids have a particular hydropathy index, indicates how hydrophobic they are Hydropathy plots can be utilized to predict membrane spanning domains in proteins. Tyr and Trp residues are often found at the ends of transmembrane α-helices because of their dual hydrophobic and hydrophilic properties. They can participate in hydrophobic interactions with the fatty acid acyl side chains and they can H-Bond with the polar head groups of the lipids in the bilayer. abv line or positive = hydrophbic below line or neg =- hydrophilic |
|
|
G-protein coupled receptors
|
targets of approximately 30% of all known drugs adopt the same overall fold as bacteriorhodopsin
bacteriorhodospin from halobacteria - 7 helices traverse bilayer - found in high salt enviro - salt lakes retinal inside- light sensitive - isomerization - captures light turns it into a signal -causes conformational change |
|
|
beta sheet and integral membrane protein
|
makes beta barrel to span lipid bilayer
beta barrel consists of 20 or more beta strands that line a cylinder only a7-9 residue beta strand is req b/c it extends more than a helix req to have hydrophobic aa at every other residue to interact w/side chains of lipid bilayer contains porin |
|
|
peripheral membrane protein
|
associates with the membranes by H-bonding and forming electrostatic interactions w/integral membrane proteins and the polar head groups of the lipid component of the bilayer
peripherial proteins can be dislodged from the bilayer w/high salt, changes in pH (alkaline carbonate) compared to the removal of integral membrane proteins from bilayer these methods are relatively gentle |
|
|
lipid anchored proteins
|
some proteins can be anchored to membranes through a covalent linckage to lipid
lipids which proteins attach include: 1. long chain fatty acids - typically attach proteins to the inner face of the plasma membrane palmitoyl groups are generally attached through a thioester linkage to a Cys residue myristoyl groups - generally attached to a N-terminal Gly residue 2. isoprenoids - ex - farnexyl and granylgeranyl groups 15-29 C that are attached to a C term Cys residue through a thioether linkage The C-term carboxylate of the Cys residue is methylated in this linkage Methylation prevents electrostatic repulsions btw the negatively charged head groups of the membrane and a neg charged carboxylate ex - Ras 3. Glycosylphosphatidylinositol (GPI) - anchors proteins to the outer extracellular face of a cell - where the other 2 types connect to the cytosolic face - the anchors are derivatives of phosphatidylinositol that has been covalently modified w/a short oligosaccharide and phosphoethanolamine the protein is linked to GPI moiety through an amide bond btw the phosphoethanolamine and its C-terminus strength of interaction btw the bilayer and the lipid anchor is relatively sml compared to the strength of the interaction btw an integral membrane protein and the bilayer other interactions (ionic or H-bonding can strengthen the rxn |
|
|
action of lipids when mixed with water
|
when amphipathic lipids are mixed w/water 3 diff agregates can form - micelles, bilayers , and liposomes
- one that forms is dependent on the types of lipid present - aggregate to minimize the amt of hydrophobic surface area that interacts w/ water minimizing the amt of ordered water (inc entropy) bilayer formation is favored when the cross-sectional areas of the head group and acyl side chains are similarly sized liposome formation occurs to minimize interactions btw water and the exposed hydrophobic edges of the bilayer |
|
|
Fluid Mosaic Model
|
phospholipids form a bilayer w/the polar regions oriented to the ext surface and hydrophobic tail regions are oriented to the int bilayer
polar head groups interact w/aqueous phase on either side of the membrane interal membrane proteins interact w/the bilayer through complementary interactions organization is asymetric individual lipids and proteins can diffuse laterally through the membrane |
|
|
temperature effect on lipid bilayer
|
low temp = bilayer semisolid gelatin-like state caused by an increase in the thermal motion of the acyl side chains
- rotation about their C-C bonds |
|
|
saturated vs unsaturated effects on membranes
|
saturated - favor semi-solid gelatin like state
unsaturated favor fluid state bc the kinks in the side chains of unsaturated acyl chains disfavor close packing of the chains |
|
|
cholesterol effects on membranes
|
high levels = reduction in overall fluidity of bilayer close to the membrane surface b/c cholesterol packs closely to the acyl chains and restricts their motion = semisolid gelatin like state
|
|
|
lipid flip flop
|
aka transverse exchange of lipid
in the absence of a catalysst lipid flip-flop is slow (t1/2~days) where as lateral and rotational diffusion occurs w/in seconds slpw b/c penetration of the hydrophilic headgroup into the hydrophobic tail region of the bilayer is strongly disfavored enzymes called flippases can catalyze the mvmt of a lipid from one side of the membrane to the other - imp for maintaining the asymmetric nature of the lipids in the bilayer |
|
|
fundamental properties of biological membranes and simple lipid bilayers
|
1. ability to self assemble
2. ability to self-seal 3. rapid rotational diffusion of lipid components 4, rapid lateral diffusion of lipid components 5. very slow transverse exchange of lipid components 6. impermeable to most polar or charged solutes 7. abt 3nm in thickness |
|
|
what can cross a membrane unassisted?
|
a few nonpolar solutes O2, CO2 and some lipid-soluble substances
|
|
|
passive transport
|
solutes diffuse down a concentration or electrochemical gradient across a membrane
|
|
|
active transport
|
cmpds pumped against conc or electrochemical gradient - E comes from ATP hydrolysis or supplied by the mvmt of another cmpd down its electrochem gradient
2 types of active transport 1. prmary active - couple the transport of a solute to a chem rxn 2. secondary active - couple the uphil transport of solute to the downhill transport of a different solute |
|
|
ionophores
|
bind to and mask the charge of ions to facillitate their transport across a membrane
ions can cross channels or be carred across by these sml molecules |
|
|
membrane potential
|
aka electrical gradient (Vm)
oppose the mvmt of neg charged ions into a compartment that is already neg charged but favor the mvmt of neg charged ions into a compartment w/an excess of + charge diffusion of ions across a membrane will continue until the charge across the membrane is equalized Vm=0 |
|
|
transport proteins
|
constitute a significatnt fraction of all the proteins encoded by the genome (>1000 in human genome)
2 general catagories 1. Channels a. pores b. gated channels 2. Carriers a. facilitated diffusio b. primary active transporters c. secondary active transporters |
|
|
Membrane transport-pores
|
generally allow rates of transport that are close to the limits of simple diffusion
transport through a pore is not saturable auaporins allow mvmt of water across membranes and are ex. linear on the Vo vs [S] graph |
|
|
membrane transport- gated channels
|
form a gated pore for ions that is either opened or closed in response to stimulus
ex of stimulus: 1.voltage 2.ligand 3.pressure 4. phosphorylation ex - conduction of nerve impulse along an axon depends on Na+ flowing into the axon through a voltage-gated channel that is opened by depolarization of the membrane transport through an opemn gated channel occurs at rates that are comparable to simple diffusion and it is not saturatable |
|
|
Cystic Fibrosis
|
defective gene is a ligand gated chloride channel that is regulated by phosphorylation
CFTR has a 2 transmembrane domains that come together to form a closed channel connected to these 2 domains are 2 nt (adenine) binding domains (ABD) and a regulatory domain (R) when the regulatory domain is phosphorylated by PKA a conformational change in the ABD occurs and these can now bind to ATP As ATP is hydrolyzed a second conformational changeresults in the opening of the channel and the diffusion of Cl- out of the cell and into the extracellular space - ultimately the confromation reverst and the channel closes transport through the CFTR channel occurs at a rate that is comparable to simple diffuseion - not saturatable |
|
|
carriers
|
bind their substrates w/high stereospecificity and catalyze transport at rates that are well below the limits of free diffusion
are saturable - abv a certain conc the rate of transport doesn't inc transport can be blocked by inhibitors 3 general classes of carrier proteins 1. facilitated diffusion - down a conc gradient 2. primary active - couple the transport of a solute to a chem rxn 3. secondary active - couple the uphil transport of a solute to the downhill transport of a diff solute b/c polar and charged molecules are hydrated in solution the shell of hydration must 1st be dissipated before the solute can pass through a membrane - energetically unfavorable so presents a barrier to the transport of any molecule however it is regained once the molecule is inside the membrane energy of activation in the absence of a transporter is so large for polar and charged solutes that lipid bilayers are nearly impermeable to them over biologically relevant periods of time specific integral membrane proteins (transporters) selectively permit the passage of the molecules by lowering the activation energy |
|
|
Facilitated diffusion
|
req the transported molecule to bind to a specific transporter in the mem so it can move from where it is highly conc to where its conc is lower
the transporter then undergoes a conformational change in order to enable the transported molecule to be released on the other side of the mem - fully reversible process and dependent only on the conc of the transported molecule on either side of them mem the rate of exit approaches the rate of entry once the concentrations equalize three hallmarks of this kind of passive transport are: 1. high rates of diffusion down a conc gradient 2. saturability 3. specificity |
|
|
GLUT1
|
glucose transporter in RBC - ex of a transporter that acts by facilitated diffusion
inc the rate of transport of glucose by 50,000folld over the rate of simple diffusion across the mem the rate of transport is saturatable described by the Michaelis menton eqn vo- Vmax[Sout]/Kt +[Sout] substrate refers to the glucose outside the cell and the product is the glucose inside the cell Kt=Km smlr Kt= smlr efficiency b/c diffusion occurs down a conc gradient RBCs cannot store glucose at a conc higher than the conc of glucose in the surrounding medium 12 glucose transporters are encoded in the human genome and each one posses its own unique kinetic properties, patterns of tis distribution and fn in RBCs GLUT1 fns to provide glucose for glycolysis the conc of blood glucose is approximately 3 times Kt which ensures that the transporter is almost completely saturated at norm blood glucose levels GLUT2 in the liver transports glucose out of the liver when liver glycogen is broken down to provide glucose for the blood GLUT 4 present in skeletal m and adipose tis - all cells have glucose transporters but in these 2 tis the activity of the transporter (GLUT4) is under the control of insulin - when insulin binds the GLUT4 transporters are housed in vesicles and they will fuse with membrane upon high insulin levels but when the insulin levels drop the GLUT4 transporters endocytose type 1 diabetes mvmt of GLUT$ from vesicles to the outer membrane is limited if insulin level is low - glucose utilization is impaired and blood glucose levels rise |
|
|
Chloride-bicarbonate exchanger (CBE)
|
In RBCs a transporter that opperates by facilitate diffusion
Inc the rate of transport by > a million fold this transporter facilitates the electroneutral mvmt of 2 anions one out for one in CO2 from respiring tis enters RBC by diffusion where it is converted into carbonic acid and then to bicarbonate which is then transported out of the RBC and one Cloride anion is transported into the plasma In the lungs the process is reversed and bicarbonate enters the RBC as CO2 is expired |
|
|
Na+K+-ATPase
|
is an ATP dependent cation transporter - ex of primary active transporter
accounts for 1/3 of the basal E req of humans maintains the cellular conc of Na+ at low levels and K+ at high levels uses ATP to cycle btwn 2 forms 1. high affinity for Na+ and low affinity for K+ 2. autophosphorylated on the Asp residue high affiniity for K+ and low affinity for Na+ hydrolysis of the phosphoryl group returns the transporter to its original form which has a high affinity for Na+ ions the hydrolysis one molecule of ATP to ADP + Pi results in the net mvmt of 2 Na+ out and 2 K+ into the cell |
|
|
Ouabain
|
a steroid derivative (Somali word for arrow poison) that inhibits the Na+K+-ATPase by preferentially binding to the extracellular side of the pump and locks 2 Na+ ions in the pump
inhibits conformational change and prevents ion transport this along w/digitoxigenin are active ingredients of digitalis - a drug used to treat CHF - raises the intracellular conc of Na+ and thereby activates a Na+CA2+ antiporter in cardiac m the increased influx of Ca2+ through this second pump strengthens the contractions of the heart |
|
|
Secondary active transport
|
ion gradients formed by primary active transport can provide E needed for this
Na+-glucose symporteres = good ex - glucose is transported against conc gradient by coupling its transport to the transport of Na+ down its electrochemical gradient 2 Na+ ions plus a single glucose molecule bind to the symporter and are transported into the epithelial cell the Na+-glucose symproter can transport glucose into the cell until intracellular conc of glucose is 9,000 times the intestinal conc of glucose |
|
|
Valinomycin
|
an ionophore
12 residue cyclic peptide that forms a complex w/a single K+ ion 6 carbonyl O2 coordinate to the K+ ion and neutralize its charge the hydrophobic character of this cyclic peptide allows it to diffuse across membranes K+ is transported down its conc gradient and in so doing collapses the electrochemical pot of the cell disrupting the secondary transport process thus valinomycin can be used as an antibiotic to disrupt transport process in microorganisms |
|
|
bioenergetics overview
|
catabolic pathways aka degradative pathways
uses chem E in C-C and C-H bonds in metabolic fuels to generate ATP and the elctron carriers NADH, NADPH, and FADH2 which are used to produce ATP these E carriers are used in anabolic (synthetic pathways to synthesize biological molecules hydrolysis of ATP to ADP provides the source of E to perform most of the work required in the cell bioenergetics is the quantitative study of cellular E transformations Gibbs free E indicates the difference in E levels btw reactants and products of a rxn |
|
|
laws of thermodynamics
|
1. the conservation of E: for any physical or chem change the total amt of E in the universe remains constant; E may change form or it may be transported from one region to another; but it cannot be created or destroyed
2nd law - universe tends towards increasing disorder: in all natural processes the entropy of the universe inc while biological org can create order they do not break the second law of thermodynamics |
|
|
Gibbs Free Energy G
|
amt of E capable of doing work under a rxn at constant temp and pressure
|
|
|
Free E change (delta G)
|
the chnage in free E
neg for a a exergonic rxn and pos for an endergonic |
|
|
delta G degree
|
the standard free energy change for a rxn startig at 1M conc of substrates and products, 25 deg C, 1 atm of gas [H+]=1M(pH=0) for reactions using H+
|
|
|
Delta G' knot
|
standard free E change (1M conc of substrates and products) at 25 degrees C, pH 7 [H2O] = 55.5M; [Mg2+]=1mM only for rxns requiring Mg2+
|
|
|
enthalpy
|
H
the heat content of the reacting system |
|
|
delta H
|
change in enthalpy
neg - exothermic positive - endothermic |
|
|
delta S
|
change in entropy
a measure of the disorder or randomness of a system when the products of a rxn are less complex than the substrates delta S is positive value bc the rxn has resulted in a gain in entropy |
|
|
K'eq
|
the eq constant at 25 deg C
pH=7 [H2O]=55.5M [Mg2+]=1mM (only for rxn requiring Mg2+ |
|
|
E' knot
|
reduction potential
|
|
|
equilibrium constant
|
A+B<=>C+D
Keq= [C][D]/[A][B] understandard condintions the same except you write K eq |
|
|
standard free E change of the rxn
|
delta G' knot is the driving force for a rxn starting under standard cond to proceed to eq
delta G' knot= -RTlnK'eq |
|
|
Delta G' knot and K'eq comparison
|
k'eq >1 then delta G is neg so the rxn proceeds forward
k'eq = 1 then delta G = 0 so the rxn is at eq k'eq<1 then delta G is positive so the rxn proceeds in reverse direction delta G' knot is not only indicative of the direction but how far the rxn must proceed to reach eq when under standard cond also - delta G = exergonic + delta G = endergonic |
|
|
Phosphoglucomutase
|
catalyzes rxn
forward rxn G6P to G1P is involved in the conversion of glucose to glycogen (glycogenesis) the reverse rxn is involved in converting glycogen into glucose-6-phosphate - a glycolytic pathway intermediate the delta G for forward rxn is +1.6 kcal/mol for reverse it is -1.6 kcal/mol |
|
|
delta G' knot for reverse rxn
|
is the same value but opposite sign
|
|
|
consecutive steps in a rxn and gibbs free E
|
additive delt G' knot
so thermodynamically unfavorable rxn can be coupled to a thermodynamically favorable rxn to drive the overall rxn in the forward direction + gibbs free energy - unfaborable under standard cond |
|
|
if thermodynamically unfavorable under standard cond
|
can be made favorable by coupling to a thermodynamically favorable rxn like hydrolysis of ATP
ex- coupling hexokinase catalyzed rxn of glucose => glucose 6 phosphate which the reverse is unfavorable in a cell so that once it is phosphorylated it is commeted to the pathway |
|
|
GLycogenesis
|
ex of an anabolic pathway that req ATP and UTP for bond making rxns
2 molecules of ATP equivalents are req to store one molecule of glucose in glycogen 1st step - glucose enters cell by facilitated diffusion 2nd step - glucose is phosphorylated to G6P by hexokinase 4. phosphoglucomutase converts G6P into G1P while delta G' knot for tis step is + and unfavorable under standard cond other factors will make it favorable later 5th step GIP is converted to an activated pathway intermediate - called UTP-glucose neg charge on phosphate on G1P acts as a nucleophile and attacks the alpha-phosphate of UTP displacing pyrophosphate PP (delta G = - 10.9) the rxn is pulled forward by the hydrolysis of pyrophosphate to 2P by inorganic pyrophosphatase (delta G' knot = -4.6 kcal//mol) for step 5 = -10.9 in the 7th step - formation of a glycosidic bond in glycogen is derived from the cleavage of glucosyl phosphate bond in UDP glucose |
|
|
Standard free E under physiological cond
|
changes only indicate the available amt of free E under standard cond
but "standard cond" are not found in a cell Alt free E expression must be used to describe the E changes that occur under physiological cond = delta G and is not constant = actual free E change for a given rxn it is the fn of the actual reactant and product conc and prevailing temp delta G for rxn proceeding spontaneoulsy towards equilibrium will always be negative - becomes less neg as the rxn proceeds and is equal to 0 at eq rxns that have a + delta G' knot can be driven in the forward direction by 1. inc the concentration of the substrate 2. decreasing the concentration of the product |
|
|
delta G related to delta G' knot
|
delta G = delta G' knot + RTln products/reactants
delta G NOT delta G' knot indicates the spontaneity of a rxn (-) |
|
|
spontaneous rxn
|
can occur even if delta G' knot is + as long as delta G is neg
this occurs when the absolute value of RTlnproducts/reactants is larger than deltaG' not and neg |
|
|
Why ATP
|
cont regeneration of ATP from ADP critical for life
process called ATP/ADP cycle the E to regenerate ATP comes from oxidation of metabolic fuels (C-C and C-H) hydrolysis of ATP and AMP plus 2Pi is highly exergonic Use of ATP and other E carriers is a common strategy used by org to carry out thermodynamically unfavorable rxns hydrolysis of ATP is highly exergonic b/c 1. charge searation upon hydrolysis relieves bond strain due to electrostatic repulsions between the beta and gamma phosphates 2. Pi is stabilized by formation of a resonance hybrid in which each of the 4 P-O bonds has the same degree of double bond character 3. ADP2- immediately ionizes releasing a proton to a solution that has a comparatively low conc of protons thermodynamically favorable - kinetically slow so ATP is a stable cmpd standard E of ATP = -7.3 kcal/mol actual free E = -12-15 due to various conc of ATP and its diff products in the cell |
|
|
high E bonds
|
ex GTP CTP and UTP are E equivalent to ATP and can be synthesized from ATP by nucleoside diphosphokinase and nucleoside monophosphokinase
ATP + UDP <=> UTP+ ADP adenylate kinase is nucleoside monophosphokinase that transfers a phosphate from one ADP molecule to another ADP to form ATP and AMP - a bond whose delta G' knot of hydrolysis is similar to or greather than the delta G' knot of hydrolysis of ATP to ADP and Pi 7.3 kcal/mol several other thioester containing cmpds have larger free E of hydrolysis than ATP ex- 1,3 bisphosphoglycerate, phosphoenolpyruvate (PEP), creatine phosphate, and acetyl-CoA some activated intermediates like UDP-glucose |
|
|
high energy bonds have neg free energys of hydrolysis b/c
|
1. charge separation upon hydrolysis relieves bond strain due to electrostatic repulsions btw neighboring neg charged groups
2. products are stabilized by ionization 3. products are stabilized by resonance stabilization 4. products are stabilized by isomerization or tautomerization |
|
|
PEP
|
ex of high E bond and a glycolysis intermediate
charge separation upon hydrolysis relieves bond strain due to electrostatic repulsions btw neighboring neg charged groups products are stabilized by isomerization/tautomerization, resonance stabilization, but not by ionization |
|
|
Glucose -6-phosphate
|
ex of a high E bond
considered low E cmpd bc hydrolysis of the phosphate group does not lead to stabilization of the product glucose by isomerization ionization or charge separation |
|
|
high E cmpds
|
can transpher phosphoryl groups to low E cmpds
ex - direct donation of a phosphoryl group from PEP to ADP to produce ATP is thermodynamically possible |
|
|
fuel oxidation
|
primary source of E for body
C-C and C-H bonds produces reduced coenzymes NADH and FADH2 which are intermediaris in transforming E in the chem bonds found in fuel to ATP E= om these reduced cmpds are ultimately transferred to O2 in the mitochondrial ETC producing an electrochem gradient of protons across the inner mito membrane that is used to regenerate ATP from ADP and Pi in a process called "oxidative phosphorylation" |
|
|
oxidative-reduction rxns
|
always involve
1. e- donor 2. e- acceptor fuel metabolism fuel donates while NAD+ and FAD accepts => NADH and FAD NAD+ generally accepts 2 e- in the form of hydride ions from alcohols and aldehydes FAD accepts 2 e- as H atoms (donated from seprate atoms (during formation of double bond) reduced coenzymes donate their E- to the ETC and are reozidized to NAD+ and FAD e- flow btw two chemical species based on differences in their affinity for e- similar to the manner in which e- flow through an electrical wire |
|
|
4 diff ways e- are transfered from e- donor to e- acceptor in biological sys
|
1. directly as e-
Fe2+ + Cu 2+ <=> Fe 3+ + Cu+ 2. As H atoms AH2 <=> A + 2e- + 2 H+ 3. As a hydride ion 4. through direct combination w/O R-CH3 + 1/2O2 -> R-CH2OH |
|
|
NAD and NADH
|
consists of 2 nt linked together through a phosphoanhydride bond
NADP+ is the phosphorylated analog of NAD+ NAD+ and NADP+ are water soluble e- acceptors that act as coenzymes for a number of enzymes that catalyze redox rxns the nicotinamide ring is synthesized from niacini niacin deficiencies common in chronic alcoholics cause Pellagra readily diffusible and accept e- in the hydride ion as oxidation of ethanol to acetaldehyde |
|
|
FAD and FADH2
|
consist of an isoalloxazine ring linked through a phosphoanhydride bond to adenine nt
derived fromo riboflavin typically remain either tightly or cofvalently attached to enzymes isoalloxazine ring can accept 1 H atom to form FADH+ (semiquinone) OR it can accept 2 H to form fully reduced FADH2 standard reduction pot for FAd is dependent on the protein that it is assoc w/ |
|
|
oxidizing agent
|
e- acceptor
|
|
|
reducing agen
|
e- donor
|
|
|
redox rxn
|
2 half rxns each w/its own standard reduction pot E' knot
standard cond are 1M of both the oxidized and reduced species E' knot is a measure in volts of the E change when that cmpd accepts e- (becomes reduced) cmpds w/+ red. pot. have the highest affinity for the e= whereas those w/more neg red. pot have more E available for ATP generation when they pass e- to O2 O2 has the larges positive E' knot and is the best e- acceptor e- tend to flow towards the half rxn w/ more + delta E' knot and greater E' knot |
|
|
delta G' knot related to delta E' knot
|
directly proportional
Delta G' knot = nFdelta E' knot n= # of e- transfered F = faraday constand 23 kcal/V*mol |
|
|
caloric value of food and oxidation state
|
directly related
highly oxidized foods (carbs) provide less E than highly reduced foods b/c oxidized foods like glucose have fewer C-H and C-C bonds from which donate e- note that the caloric value of a food is only applicable to humans if we posses the enzymes to oxidize it ex - cholesterol contains many C-H and C-C bonds but humans lack the enzymes to transfer the e- in these bonds to NAD+ or FAD so caloric content of cholesterol for humans is 0 |
|
|
CAC summary
|
during each durn acetyl CoA donates acetyl group to oxaloacetate, a 4 C cmpd to generate citrate -> i
socitrate - oxidatively decarboxylated to form alph-ketoglutarate leading to the generation of NADH alpha-hetoglutarate reacts w/CoASH to form succynyl- CoA w/ resultant loss of CO2 and the formation of NADH substrate level phosphorylation generates succinate, GTP and CoASH succinate is then converted to oxaloacetate in 4 steps that together generate 1 molecule each of FADH2 and NADH |
|
|
citrate synthesis
|
citrate synthase catalyzes the condensation of oxaloacetate w/acetyl group of acetyl CoA to form citrate
citrate synthase catalyzes formaiton of bond btw methyl C of acetyl CoA and the carbonyl C of oxaloacetate favorable large neg delta G' knot of thioester hydrolysis |
|
|
formation of isocitrate
|
via cis-aconitate
aconitase caatalyses the reversible rearrange of citrate to isocitrate rxn occurs in 2 steps and invlolves formation of a stable intermediate that does not dissociate from the enzyme active site at eq isocitrate is 10% of the citrate and the forward rxn under standard cond is energetically unfavorable delta G' knot = +1.5 kcal/mol but b/c it is readily generated b/c its conc is maintained low level by participation in the next step of the cycle -effectively lowers its steady state conc and drives the rxn forward |
|
|
formation of alpha-ketoglutarate
|
isocitrate dehydrogenase catalyzes the oxidative decarb. of isocitrate to form this , converts 6C cmpd to a 5 C cmpd
in the 1st step the alcohol is oxidized to a ketone w/C-H e- from NAD+ as a hydride anion to form NADH. the proton from the OH group dissociates into water ultimately leads to the loss of CO2 - not from original acetyl group and 1st rxn to produce NADH |
|
|
formation of succinyl-CoA and CO2
|
second oxidative decarboxylation step in CAC
rxn is catalyzed by alph-ketoglutarate dehydrogenase complex that has 2 subunits E1 E2 E3 that are structurally related to subunits of the pyruvate dehydrogenase complex alpha ketoglutarate dehydrogenase complex also uses 5 coenzymes: thiamine pyrophosphate, lipoate, CoASH, FAD, NAD+ 2 enzymes use similar rxn mechanisms ang both generate a reactive thioester 2nd rxn that generates a NADH and CO2 released not from original acetyl group |
|
|
formation of L-Malate
|
fumarase catalyzes the reversible hydration of fumarate
a carbanion is formed during the transition state fumarase is highly stereospecific and will only catalyze the hydration of the trans isomer only fumarate is a substrate and not maleat the cis isomer of furmate |
|
|
formation of oxaloacetate
|
last rxn of CAC L-malate dehydrogenase catalyzes the NAD+ dependent oxidation of oxaloacetate
delta G'knot for the rxn indicates that under standard cond the eq lies far to the left however in cells oxaloacetate is maintained at low levels by the action of citrate synthase therefore the rxn is favorable under physiological conditions this rxn produces a third molecule of NADH |
|
|
energetics of the CAC
|
a single turn results in the production of
3 molecules of NADH - 3x2.5= 7.5 molecules of ATP 1 molecule of GTP = 1 molecule of ATP 1 molecule of FADH2 = 1.5 molecules of ATP overall net neg delta G'knot=-13 kcal/mol overall E favorable although some individual rxns are unfavorable highly efficient generator of E total amt of E in an acetyl group is ~ 228 kcal/mol (determined by measuring E released from the total combustion of an acetyl group) this compares to the products of the CAC (NADH, FADH2, GTP) that together contain a total of ~207 kcal/mol of E - thus CAC conserves ~90% of the E available from complete oxidation of an acetyl group |
|
|
reversible and irreversible rxn of CAC
|
rxns catalyzed by citrate synthase, isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase have large neg delta G'knot
these rxn are essentially irreversible under physiological cond b/c 1. product conc is norm low 2. enzymes are poor catalysts for reverse rxn rxn catalyzed by aconitase and malate dehydrogenase have + deltaG'knot and are thermodynamically and kinetically reversible - forward and reverse rxn catalyzed rapidly for the aconitase rxn citrate is typically higher than that of isocitrate the accumulation of citrate is imp b/c it can be transported out of the mitochondrion into the cytosol and used in faty acid and cholesterol biosynthesis formation of oxaloacetate from malate by malate dehydrogenase is driven in part by NADH/NAD+ ration. In liver during fasting ther is a net flux of oxaloacetate towards malate which can then be trasported out to the cytosol to be used as a gluconeogenic precursor this occurs b/c when fats are mobilized the high level of fatty acid oxidation casue inc in NADH levels |
|
|
regulation of CAC
|
regulated by several allosteric effectors including the E carriers ATP, ADP, NAD+, NADH, and Ca2+ - acetyl CoA needs only to be oxidaixed as fast as NADH and FADH2 are oxidized by the ETC to generate ATP
ATP levels and NADH/NAD+ ratio are major factors that control the flux w/in mitochondrion the total adenine nt pool is relatively constant (ATP,AMP,ADP) inc in ATP use will dec the adenine nt levels and inc the conc of ADP similary the total NAD pool is constant increased oxidation of NADH raises NAD+ levels which will inc the rate of rxns that lead to the production of NADH alll factors are coordinated to maintain constant levels ofATP w/in cell ATP homeostasis |
|
|
4 major sites of regulation of CAC
|
1. pyruvate dehydrogenase complex: regulation at this step helps to control the production of acetyl Co-A
2. Citrate synthase - regulation here helps control the entry of acetyl CoA into the cycle 3. isocitrate dehydrogenase 4. alpha ketoglutarate dehydrogenase 3&4 are rate limiting steps in CAC due to kinetic irreversibility |
|
|
isocitrate dehydrogenase
|
rate limiting step
2 active sites and is composed of 8 subunits isocitrate NAD+, and NADH bind at active site wheras ADP and Ca2+ are allosteric activators that bind at diff sites exhibits positive cooperativity in presence of ADP the subunits are in the active conformation and the Km for isocitrate dec - the affinity ofthe enzyme for isocitrate is inc the effect of ADP on isocitrate dehydrogenase is not an all or nothing respose - extent of activation depends on the [ADP] can stimulate up to a ~6 fold inc in the velocity NADH is a product inhibitor of isocitrate dehydrogenase and as the conc of NADH inc the rate of the enzyme catalyzed rrxn dec |
|
|
alpha-ketoglutarate dehydrogenase
|
subject to product inhibition by both succinyl CoA and NADH
b/c the enzyme is inhibited by NADH, its rate is controlled by the rate of NADH oxidation in the ETC the activity of this enzyme is up-regulated in the presence of Ca2+ Ca2+ release occurs during m contraction and since m contraction req high level of ATP hydrolysis this mode of regulation is likely imp for maintaining E status of cell |
|
|
anaplerotic rxns
|
rxns that replenish CAC
req to regenerate oxaloacetate - imp b/c in absent acetyl-CoA cannot be oxidized to compensate for the use of CAC intermediates in biosynthetic pathways, cells must synthesize enough 4-C intermediates this ensures that conc of CC intremediates remain nearly constant |
|
|
to return
|
العَوْدة إلئ / عَادَ إلئ / يَعُودُ إلئ
|
|
|
AA degradation an anaplerotic rxn of CAC
|
oxidation of several AA leads to formation of 4 and 5 C CAC intermediates
Ala and Ser can be converted into pyruvate and their C used as a source for synthesis of oxaloacetate in all tiss except liver oxidation of Ile and Val leads to the formation of succinyl CoA in the liver, odd chain (but not even chain) fatty acids, Met, and thymine serve as precursors for propionul-CoA which can be converted to succinyl-CoA by propionyl-CoA in most tis Gln is taken up from the blood, converted to Glu and then further converted to alpha ketoglutarate by glutamate dehydrogenase additionally oxaloacetate and alpha-ketoglutarate can be synthesized from Asp and Glu respectively in simple transamination rxns |
