When the antibiotic valinomycin is added to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases.
Valinomycin acts as an uncoupler.
In actively respiring mitochondria, H+ are pumped into the intermembrane space, creating a proton gradient and pH difference across the inner mitochondrial membrane. The proton gradient is utilized for ATP synthesis.
Natu
When O2 is added to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the O2 is used up, and accumulation of lactate ceases. This effect, first observed by Louis Pasteur in the 1860s, is
Addition of oxygen to an anaerobic suspension allows cells to regenerate NAD+ required for glycolysis via oxidative phosphorylation instead of fermentation. Oxidative phosphorylation also produces more ATP per glucose molecule as compared to fermentation.
In the presence of saturating amounts of oxaloacetate, the activity of citrate synthase from pig heart tissue shows a sigmoid dependence on the concentration of acetyl- CoA, as shown in the graph. When succinyl-CoA is added, the curve shifts to the right
Citric acid cycle intermediates are used at steady state in a normally respiring cell. Accumulation of an intermediate of this cycle suggests reduced metabolite flux. Thus, accumulation of Succinyl-CoA suggests reduced flux through citric acid cycle.
Citr
The effect of ATP on the allosteric enzyme PFK-1 is shown below. For a given concentration of fructose 6-phosphate, the PFK-1 activity increases with increasing concentrations of ATP, but a point is reached beyond which increasing the concentration of ATP
ATP is a substrate as well as allosteric inhibitor of PFK-1.
Although ATP is one of the substrates of PFK-1, high concentration of ATP saturates the catalytic site. This increases the binding of ATP to the allosteric site, inhibiting the PFK-1.
Increased
The conversion of pyruvate to acetyl-CoA is negatively regulated by two molecules that do not negatively regulate any step in the citric acid cycle. Identify these molecules, and discuss the physiological reasons that they are not negative regulators of c
Fatty acids and acetyl-CoA negatively regulate production of acetyl-CoA but not any citric acid cycle enzymes. Fatty acids are metabolized to form acetyl-CoA. Both fatty acids and acetyl-CoA signal high energy availability and a need to convert that energ
Describe the chemiosmotic theory for coupling oxidation to phosphorylation in mitochondria.
Electron flow through membrane-bound electron transport chain proteins helps generate energy to actively transport protons across the inner mitochondrial membrane creating a proton gradient (proton motive force).
The proton diffuses back across the inner
Explain why anything that makes the mitochondrial membrane leaky stops ATP synthesis in the mitochondria.
Electron flow through membrane-bound electron transport chain proteins helps generate energy to actively transport protons across the inner mitochondrial membrane creating a proton gradient (proton motive force). The proton diffuses back across the inner
A) Write all the reactions for the conversion of glucose to glycogen using chemical names rather than structures.
B) Show the participation of all the cofactors involved and the names of the specific enzymes for each step.
Glucose + ATP glucose 6-phosphate + ADP Glucose 6-phosphateglucose 1-phosphate
Glucose 1-phosphate + UTP UDP-glucose + PPi UDP-glucose + glycogen (n) glycogen (n+1) + UDP
Hexokinase; Mg 2+
Phosphoglucomutase
UDP-glucose pyrophosphorylase
Glycogen synthase
What is the sequence of reduction reactions catalyzed by the ?-ketoglutarate dehydrogenase complex?
Lipoyllysine (oxidized) ? acyl lipoyllysine followed by FAD ? FADH2 ending with NAD+ ? NADH
Given the structures of oxaloacetate and acetyl- CoA below, which numbered carbon atoms are lost as CO2 molecules during the citric acid cycle?
1 and 4
Given a single acetyl-CoA molecule, how many ATP molecules will result from the combination of 1 turn through the citric acid cycle and the electron transport chain/oxidative phosphorylation?
10
Which one of the following types of mechanisms is not known to play a role in the reversible alteration of enzyme activity?
Activation by cleavage of an inactive
zymogen
Antimycin A blocks electron transfer from ubiquinol to cytochromes b & c1. If intact mitochondria were incubated with antimycin A, excess NADH and adequate supply of O2, which of the following would be found in the oxidized state?
Cytochrome a3
In the reoxidation of QH2 by purified ubiquinone- cytochrome c reductase (complex III) from heart muscle, the overall stoichiometry of the reaction requires 2 molecules of cytochrome c per molecule of QH2 because:
Cytochrome c is a one electron acceptor, whereas QH2 is a two electron donor
Select the FALSE statement about the regulation of glycogen phosphorylase.
a. Glucose binds to the glycogen
phosphorylase a allosteric site, exposing the phosphorylation sites to the phosphatase to promote dephosphorylation
*****b. Epinephrine and glucagon stimulate activation of glycogen phosphorylase by binding to an allosteri
Upon the addition of 2,4-dinitrophenol (DNP) to a suspension of mitochondria carrying out oxidative phosphorylation, protons are released into the _________ and __________
d. Matrix; oxygen consumption increases
Which one of the following is not a characteristic of phosphorylase a phosphatase
or phosphoprotein phosphatase-1 (PP1)?
PP1 is phosphorylated by glycogen synthase kinase-3 (GSK3)
Which one of the following steps or components in electron transport is directly inhibited by Amytal?
NADH ? coenzyme Q
The aconitase active site has an iron-sulfur center. Select the contribution of the iron-sulfur cluster that allows the elimination reaction to occur by the E2 mechanism.
An iron atom in the cluster chelates the
hydroxyl group, making it a good leaving
group as required in the E2 mechanism
Which of the following is NOT water soluble and is NOT a mobile electron carrier?
*****a. Cytochrome a3 Fe3+/Fe2+
b. Cytochrome c Fe3+/Fe2+
c. 1?2 O2/H2O
d. Coenzyme Q/QH2
e. NAD+/NADH
Which of the following statements is FALSE?
a. Cytosolic NADH cannot directly permeate
the inner mitochondrial membrane
b. The malate-aspartate shuttle recreates
NADH in the membrane
****c. Cytosolic FADH2 enters the mitochondrial matrix via specialized shuttle
d. Transport of reducing equivalents
The reversible conversion of L-malate to oxaloacetate catalyzed by malate dehydrogenase has a biochemical standard free energy of +29.7 kJ/mol. What prevents the reaction from performing the reverse process (converting oxaloacetate into L-malate)?
Low concentration of oxaloacetate leading to a very low mass-action ratio and favorable free energy change under cellular conditions
Citrate synthase catalyzes the highly exergonic first step in the citric acid cycle. Which of the following explains why the biochemical standard free energy is so negative?
The high energy thioester bond of acetyl CoA is hydrolyzed during the reaction
Gluconeogenesis must use "bypass reactions" to circumvent three reactions in the glycolytic pathway that are highly exergonic and essentially irreversible. Reactions carried out by which of three of the enzymes listed must be bypassed in the gluconeogenic
d. 1,3,4
Identify the nucleophile (N), electrophile (E), and leaving group (LG) in the reaction catalyzed by glycogen synthase.
1) UDP-glucose anomeric oxygen atom
2) UDP-glucose anomeric carbon atom
3) UDP
4) Glycogen reducing end anomeric
hydroxyl group
5) Glyco
N-5;E-2;LG-3
Which of the following statements is FALSE?
a. the P:O ratio is the number of ATPs formed
in oxidative phosphorylation for each two electrons which flow through the electron transport chain to oxygen
***b. complex I, II, III, IV of the electron transport chain all transport H+ into the intermembran
Choose an effect specific to an uncoupler (unique from an ATP synthase inhibitor):
the uncoupler would increase heat production by the mitochondria
Which of the following statements about gluconeogenesis in animal cells is TRUE?
a. a rise in the cellular level of fructose 2,6
bisphosphate stimulates the rate of
gluconeogenesis
b. an animal fed a large excess of fat in the diet
will convert any fat not needed for energy production into glycogen to be stored for later use
***c. the
What is the purpose of the anaplerotic reactions?
To replenish concentrations of oxaloacetate and malate
Which of the following is true of glycogen synthase?
a. activation of the enzyme involves a phosphorylation
***b. it catalyzes addition of glucose residues to the non-reducing end of a glycogen chain by formation of (14) bonds
c. it uses glucose 6-phosphate as donor of glucose units
d. the conversion of an
Pyruvate dehydrogenase (E1 of the pyruvate dehydrogenase complex) requires which of the following coenzymes?
TPP
Glycogen phosphorylase a can be inhibited at an allosteric site by:
Glucose
The immediate product of the degradation of glycogen by phosphorylase a is
Glucose 1-phosphate
Fructose 2, 6-bisphosphate allosterically _______ the affinity of PFK-1 to _______.
Decreases; ATP & citrate
Keq for phosphofructokinase-1 (PFK-1) reaction is approximately 1000. Q for this reaction is at a steady state of 0.1. Identify the mass action ratio that will make this reaction work far from equilibrium and in forward direction under cellular conditions
[Fructose 1,6-bisphosphate][ADP] / [Fructose 6-phosphate][ATP]; exergonic
A value of 1.0 for flux control coefficient "C" indicates that:
An enzyme in a metabolic pathway solely determine the metabolite flux in the pathway
IN an allosteric enzyme, elasticity coeffecient of 1.0 indicates
Positive cooperativity
In muscle cell, insulin effects glycogen synthesis from blood glucose. Increase in synthesis of _________, increases the glucose flux towards glycogen formation.
hexokinase
Hexokinase I and II are __________ inhibited by __________. Whereas, hexokinase IV is inhibited by _____________ to a ___________.
Allosterically, Glucose 6-phosphate; reversible binding, regulatory protein
Glucose transporter, ________ is responsible for the uptake and release of glucose into the blood capillaries from hepatocytes.
GLUT 2
_________ activates _________, which favors glycolysis by ________ of _________.
Xylulose 5-phosphate; phosphoprotein phosphatase-2A; dephosphorylation; PFK- 2/FBPsse-2 complex
McArdle's disease (a glycogen storage disease) results from a defect or deficiency in one of the enzymes in glycogen breakdown. Patients with this disease suffer from painful muscle cramping from strenuous exercise, yet recover after brief rest. A deficie
Muscle glycogen phosphorylase
Glycogen phosphorylase act on non-reducing ends of glycogen. Glycogen phosphorylase can only break down glycogen until it reaches
4 glucose residues; (alpha16) branch
The only reaction in the citric acid cycle where carbon-carbon bond is formed is catalyzed by
c. Citrate synthase
Which of the following enzymes in the Krebs cycle does not use the same mechanism as pyruvate dehydrogenase?
c. aconitase
Which inhibitor and complex inhibited is correct?
a. Rotenone - Complex II
***b. Antimycin A - Complex III
c. Cyanide - Complex I
d. Oligomycin - Complex I
e. Carbon monoxide - Complex II
If you purify endoplasmic reticulum vesicles from liver cells, and add glucose 6-phosphate, what product would you expect to observe in the medium?
glucose
If acetyl-CoA was labeled on its methyl group, where would the label end up following one turn of the glyoxylate cycle?
50% on the methylene, 50% on the keto group oxaloacetate
What is the net change in number of the protons across the mitochondrial membrane for a pair of electrons transferred through complex I, III and IV respectively?
4,4,2
A cycle can be constructed with the enzymes: Pyruvate kinase (PEP to pyruvate)
Pyruvate carboxylase (pyruvate to oxaloacetate) PEP carboxylase (oxaloacetate to PEP)
What would be the net equation for one turn of this cycle?
CO2 + H2O --> HCO3- + H+
Pyruvate labeled on its methyl group is fed to an organism. In the first turn of the citric acid cycle, the label is found in _______ of -ketoglutarate and _____ of oxaloacetate.
Position 4 / half in the methylene group, half
in the -keto group
Isocitrate dehydrogenase is regulated by phosphorylation; the protein kinase/phosphatase is in turn allosterically regulated by a variety of metabolites. Which of the following compounds might be expected to have the negative regulatory effect on the prot
ATP
At the cytochrome oxidase (complex IV)
An oxygen molecule is bound and four electrons and four protons are added to make two water molecule
Which enzyme in the citric acid cycle catalyzes a substrate level phosphorylation?
Succinyl-CoA synthetase
Assume that the pH of the cytosol is 6 and the pH of the matrix of mitochondria is 7. The pH of the intermembrane space of the mitochondria is:
6
How many fewer ATPs are produced in each turn of glyoxylate cycle (starting from citrate) as opposed to the citric acid cycle? Assume 2.5 ATP/NADH and 1.5 ATP/FADH2
3.5
The reaction catalyzed by succinyl CoA synthetase is most like the reaction catalyzed by which glycolytic enzyme?
Phosphoglycerate kinase
In
the first phase of pyruvate carboxylase reaction:
Activate CO2 is generated from ATP & bicarbonate and then used to carboxylate biotin
In the pyruvate dehydrogenase complex _________ accept ______ and the ______ derived from ______.
Dihydrolipoyl transacetylase; 2 electrons;
acetyl group; pyruvate
Which of the following is correct concerning the mitochondrial ATP synthase?
When it catalyzes the ATP synthesis reaction, the G is actually close to zero.
The final electron acceptor in the reaction catalyzed by complex II is
ubiquinone
How many protons are moved across the mitochondrial inner membrane, when transferring a pair of electron form FAD to acyl- COA dehydrogenase to 1?2 O2?
6
Active glycogen phosphorylase inhibit activation of glycogen synthase by inhibiting
Phosphoprotein phosphatase 1
Succinyl CoA, an intermediate of citric acid cycle, is utilized for production of:
Prophyrins
Which of the following statements about the chemiosmotic theory is false?
a. Electrons transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane.
b. Energy is conserved as a trans-membrane pH gradient
c. Oxidative phosphorylation cannot occur in membrane fee prep
Mammals produce heat by using the endogenous uncoupling agent:
The protein thermogenin