Biochem 2 Midterm 1 Flashcards
If △G°’ > 0 how can you make a reaction occur?
1) Change the concentrations of reactants and products: cascade of reactions in biological systems controls this, downstream rxn uses previous reaction, compartmentalization allows for this
2) Couple with another exergonic reaction so that △Gnet < 0
What is the benefit of pairing two reactions?
even though one reaction is energetically favorable it may have high activation energy or have alot of requirements to occur
What is catabolism?
degradation, energy production, carbs, lipids, proteins
What is anabolism?
biosynthesis, synthesis of macromolecules, muscle contraction, active ion transport, thermogenesis
What are the high energy bonds in ATP? How many are there?
phosphoanhydride bonds, one ATP molecule has 4
What are high energy bonds?
- cleavage yields energy more negative than -25kj/mol
- not bond energy
- does not refer to the intrinsic stability of a molecule
- only refers to the stability of the cleaved product being higher than that of the reactant
What ion is important for stabilizing ATP?
- magnesium is essential for balancing out electrostatic repulsion
How do organisms save free energy?
- organisms capture free energy in high energy compounds whose subsequent breakdown is used to power endergonic reactions
- high energy compounds and reducing equivalents
Why is the hydrolysis of a phosphoanhydride bond so exergonic?
1) The resonance stabilization of a phosphoanhydride bond is less than that of its hydrolysis products
2) There is destabilizing effect of electrostatic repulsions of the charged groups of a phosphoanhydride
3) Smaller solvation energy of a phosphoanhydride compared to that of its hydrolysis products
How do catabolism and anabolism relate to redox?
Catabolism: reduced substrates to oxidized products
Anabolism: oxidized precursor to reduced products
What occurs in a redox reaction?
- electrons move between atoms
- reduction: gain electrons, oxidation number decreases
- oxidation: lose electrons, oxidation number increases
What are the oxidation rules (carbon bonds)?
C - C = 0
C - H = -1
C - (O,N,S) = +1
What are the oxidized and reduced forms of NAD+/H2
NAD+ = oxidized form NADH = reduced form
What is NADH derived from?
Niacin or vitamin B3 (or Tryptophan)
- cant make it from our diet
What is pellagra?
- rough skin caused by a lack of niacin (VB3) and tryptophan from diet
- symptoms: dementia, diarrhea, dermatitis, and death
- in mexico there are fewer cases because corn is usually cooked alkaline solution that helps release niacin from the bound state to get used by the human body
What is an enzyme that catalyzed oxidation of NADH to NAD+?
- Lactate dehydrogenase
Pyruvate + NADH + H2O -> Lactate + NAD+ OH
What are three pathways through which Acetyl CoA can be produced?
Glucose -> Pyruvate -> Acetyl CoA
Fats/Lipids -> oxidized to Acetyl CoA
Proteins -> proteinolysis to amino acids -> Acetyl CoA
What is the reverse process of glycolysis?
gluconeogenesis
How does acetyl coA store energy?
- thioester bond
- breaking this bond △G°’ = -31.5kj/mol
Why does a thioester bond have higher △G for hydrolysis than an oxygen ester?
Oxygen ester has more resonance stabilization
What is glucose catabolism called?
glycolysis
Where does glycolysis occur?
in the cytosol of most eukaryotic cells
What is the reaction of glycolysis?
Glucose + 2NAD+ + 2ADP + 2Pi –> 2 pyruvate + 2NADH +2ATP + 2H2O + 4H+
Glycolysis occurs without______
oxygen
What are the objectives of glycolysis?
- produce energy
- produce precursors for biomass (critical intermediates in biosynthesis)
Why is phosphorylation important in glycolysis?
1) when glucose becomes phosphorylated it becomes negative and gets trapped inside of the cell (phosphorylated sugar cannot leave the cell)
2) adding a phosphate group onto glucose activates/destabilizes glucose
3) phosphates form complexes with Mg2+ ions in enzyme active sites
What is a kinase?
an enzyme that transfers phosphate groups from ATP
Why is glucose used to store energy?
1) it was likely available as fuel for primitive organisms
2) has low tendency to glycate proteins
List the first 5 enzymes of glycolysis in order.
1) Hexokinase
2) Phosphoglucose Isomerase (PGI)
3) Phosphofructokinase 1 (PFK-1)
4) Aldolase
5) Triose Phosphate Isomerase
Describe the first stage of glycolysis.
- no electron transfer
- one molecule of glucose to two molecules of GAP
- 2 ATP consumed
- (energy investment stage)
Describe the thermodynamics of the hexokinase reaction. (Step 1)
- reaction of glucose + phosphate (to form G6P) is thermodynamically unfavorable
- coupled to exergonic cleavage of ATP
- overall reaction is thermodynamically favorable
What occurs when glucose binds to hexokinase? (Step 1 Glycolysis)
- glucose induces a large conformational change in hexokinase
- movement excludes water from the active site, and prevent hydrolysis of ATP
- this functions to bring the reactants together
Describe the PFK-1 reaction. (Step 3 Glycolysis)
- F6P to FBP
- PFK-1 reaction is very similar to hexokinase reaction
- PFK is an important reaction because it catalyzes one of the pathways rate-determining steps
Describe the structure of PFK-1. How is this reaction regulated? (Step 3 Glycolysis)
- PFK-1 is a tetramer with four identical subunits
- These subunits act cooperatively with each other
- R and T states
- Regulated allosterically
Describe the allosteric regulation of PFK-1. (Step 3)
- PFK-1 subunits have two conformers: R & T
- R (active) and T (inactive) bind ATP equally in the substrate site
- Only T binds ATP in the regulatory site
- When ATP is high it drives the equation to the T state
- Only R state binds substrate (F6P)
- Another Mechanism: AMP, ADP, and F2, 6 bisphosphate binding facilitates the R state (this process overrides ATP allosteric binding)
What is Tarui disease?
- Patients experience exercise induced muscle cramps and weakness and myoglobinuria, can cause hemolytic anemia
- caused by PFK-1 deficiency
What mechanism does aldolase proceed through? (Step 4 Glycolysis)
retro-aldol condensation
Describe the triose phosphate isomerase enzyme. (Step 5 Glycolysis)
- essential residues: Glu, His, Lys (Glu abstracts proton, his protonates carbonyl, lys stabilizes negative transition state)
- TIM is an alpha/beta barrel protein (TIM barrel); 8 parallel beta strands surrounded by 8 parallel alpha helices
What is unique about triose phosphate isomerase (Step 5)
- TIM is a catalytically perfect enzyme - rate is diffusion controlled
- GAP and DGAP are interconverted so efficiently that concentrations are maintained at equilibrium value; GAP consumed before DHAP by glycolytic flux
What is the enzyme in step 6 of glycolysis and how does it function?
- GAPDH couples aldehyde oxidation (exergonic reaction) to the synthesis of high energy 1,3-BPG
- GAPDH can be thought of as catalyzing two reactions
- GAPDH forms a high energy thioester intermediate
- Thiol group from cysteine residue -S-H
- NAD+ alters the chemical reactivity and polarizes decreasing the pKa of the thiol group from 8 to 3.5, making it easier for deprotonated thiol to form
What is phosphoryl transfer potential? When is it relevant?
- the amount of energy released when a phosphorylated compound transfers its phosphoryl group to water
- GAPDH produces the first “high-energy” intermediate in glycolysis
- 1,3 BPG has a greater phosphoryl transfer potential than ATP (Acyl Phosphate)
GAPDH is _______
- GAPDH is a dehydrogenase, not a kinase
- It is a double displacement reaction
What are the two types of biochemical reactions?
- single displacement reactions (sequential)
- double displacement reactions (ping-pong)
What is a single displacement reaction?
- all substrates must combine with the enzyme before a reaction occurs and products are released
What is a double displacement reaction?
- first the substrate transfers a functional group to the enzyme, then a second substrate displaces it to yield the product
Describe glycolysis in cancer.
- cancer cells grow faster - results in hypoxia
- glycolysis is 10x faster in cancer cells
- GAPDH is overexpressed in many cancers including melanoma and colon cancer
Describe Phosphoglycerate Kinase (Step 7 Glycolysis)
- has an induced fit model similar to hexokinase
- functions as a reverse kinase - reverse process is phosphorylation
- GAPDH and PGK reactions are coupled, 1,3 BPG consumption pulls GAPDH reaction forward
Describe Phosphoglycerate mutase (Step 8 Glycolysis)
- phosphate is the same group just moved to a different location on the molecule
- 2,3 BPG Enzyme Complex is formed
- PGM has a phosphorylated histidine in the active site
Describe Enolase (Step 9 Glycolysis)
- Forms complex with two Mg2+ ions
- Fluoride ions inhibit glycolysis by blocking enolase, blocks substrate binding by forming a complex with Mg2+ at the active site and preventing binding of 2PG
- Forms high energy intermediate: PEP
Describe Pyruvate Kinase (Step 10 Glycolysis)
- Couples free energy of PEP cleavage to the synthesis of ATP during the formation of pyruvate
- PK reaction requires monovalent K+ and divalent Mg2+
How is glycolysis regulated?
- the steps with the largest changes in △G are might tightly regulated
- 1, (hexokinase), 3 (PFK-1), and 10 (PK)
What are three ways a reaction may be regulated?
- metabolic effectors - changing the concentrations of metabolites (allosteric -> most rapid response, fastest) (ms)
- second messengers - hormones (seconds)
- genes - turning on and off - slowest regulation (hours)
What are the three fates of pyruvate?
- under aerobic conditions, pyruvate is completely oxidized via the citric acid cycle
- under anaerobic conditions, pyruvate must be converted to a reduced end product in order to reoxidize the NADH produced by GAPDH: reduced to lactate, or decarboxylated to ethanol
Describe homolactic fermentation.
- in muscle during vigorous activity (ATP demand is high, oxygen is limited); ATP is synthesized via anaerobic glycolysis
- lactate dehydrogenase catalyzes the oxidation of NADH by pyruvate to yield NAD+ and lactate
- this reaction is freely reversible, so pyruvate and lactate concentrations are readily equilibrated
Describe alcoholic fermentation (yeast).
- pyruvate is converted to ethanol and carbon dioxide
- occurs in two steps catalyzed by pyruvate decarboxylase and yeast alcohol dehydrogenase
What is required for pyruvate decarboxylase to function?
- contains thiamine pyrophosphate (TPP) which is synthesized from thiamin (vitamin B1)
- TPP is used to diffuse negative charge formed during the reaction
What is the pentose phosphate pathway?
- generates NADPH
- NAD+/NADH favors metabolic oxidation
- NADP+/NADPH favors reductive biosynthesis
What is gluconeogenesis?
- synthesizing glucose from pyruvate
- mainly occurs in liver
- closely regulates blood sugar
- liver makes new glucose and releases it into the blood
- occurs in mitochondria, cytosol, and ER
What two enzymes catalyze the first step of gluconeogenesis (reverse of step 10 of glycolysis)?
pyruvate carboxylase - (oxyloacetate) -PEPCK
What does pyruvate carboxylase require and how is it regulated?
- requires biotin cofactor
- activated allosterically by acetyl CoA
What are the two routes used in gluconeogenesis?
- may start from lactate or pyruvate
What are the 3rd and 4th enzymes that catalyze gluconeogenesis?
- Fructose 1,6 bisphosphatase and glucose-6-phosphatase
Glycolysis and gluconeogenesis are ____
independently regulated
Why is hyperglycemia bad?
- organs dont have the ability to restrict uptake of glucose (peripheral nerves, kidneys, and retina) so they act as sponges and absorb excess glucose
- over absorption of glucose generates superoxides that activate poly ADP-ribose polymerase (PARP) which ribosylates GAPDH
- GAPDH - cysteine residue, which forms thioester bond is damaged in diabetes and inhibits glycolysis
What is the function of the Schiff base in the aldolase reaction?
Because of its positive charge, protonated Schiff base is a better electron withdrawing group than carbonyl oxygen
What is the role of TPP in pyruvate decarboxylase?
- TPP is an electron sink of the transition site, it diffused negative charge formed during the reaction
- synthesized from vitamin B1
How does the liver buffer blood glucose level?
- Cells in pancreas release hormones
- Alpha cells -> glucagon -> increase [CAMP]+ -> protein kinase A
- Beta cells -> insulin -> phosphoprotein phosphatase
How is pyruvate carboxylase regulated?
- activated allosterically by acetyl CoA
How is PEPCK regulated?
- regulation is more transcriptionally controlled
- insulin increases activity
- glucagon decreases activity
Explain F26BP.
- potent activator of PFK
- inhibitor of FBPase
- synthesized by PFK2 - activated by PP1
- degraded by FBPase2 - activated by PKA
Which reaction is F26BP at play in?
3rd reaction of glycolysis - activates PFK
What is glycogen?
storage form of glucose as a polymer
What does glycogen level indicate?
sugar storage
How do liver and muscle buffer blood sugar?
- liver - uptake and release
- muscle - uptake only
Why do we have glycogen?
1) Glycogenolysis is faster than fatty acid release and degradation
2) fat cant be metabolized into glucose, necessary for brain
3) fats cannot be used without O2
What is glycogen structure?
- a (1-4) linear
- a (1-6) branched
What is the purpose of branching of glycogen?
1) increases solubility
2) creates more sites of action for enzymes
Which glycogen enzymes are faster?
a (1-4) (linear) are faster than a (1-6) (branched)
What are the three glycogen break down enzymes?
1) glycogen phosphorylase
2) debranching enzyme
3) Phosphoglucomutase
What are problems with glycogen synthase?
- need branching - branching enzyme
- need primer - glycogenin
What is glycogenin and what does it do?
- glycogenin attaches a glucose residue donated by UDPG to the OH group of its Tyr 194
- then it extends the chain by up to 7 residues to form a small glycogen so that glycogen synthase can commence synthesis
What molecules are responsible for regulating the flux of glycogen synthesis and glycogenolysis?
- glycogen phosphorylase
- glycogen synthase
What are PTMS?
post translational modification
Where do PTMs occur?
Ser, Thr, Tyr
What does a protein kinase do?
add Pi from ATP to a molecule
What does a protein phosphatase do?
removes a Pi from a molecule (H20) to Pi
How can phosphorylation affect a protein?
1) conformational change
2) affects electrostatic interactions
What enzymes activate and deactivate glycogen phosphorylase and glycogen synthase?
phosphorylase kinase and phosphoprotein phosphatase
What activates phosphorylase kinase?
- maximally activated by Ca2+ (muscle contraction)
- phosphorylation by PKA
What activates PKA?
cAMP
The citric acid cycle is a _____ process
amphibolic (catabolic and anabolic functions)
What are the catabolic and anabolic processes of TCA?
- catabolic - production of reducing equivalents (NADH and GTP)
- anabolic - production of metabolites important for synthesis
What is catapleurosis?
pull out metabolites to make other things
What is anapleurosis?
refill metabolites into the TCA
What must happen before pyruvate can enter the TCA?
pyruvate dehydrogenase complex turns pyruvate into Acetyl-CoA
In addition to metabolic effectors, second messengers, and genes what else regulates flux through glycolysis and gluconeogenesis?
Substrate cycling
What is substrate cycling?
for non-equilibrium reactions 2 different enzymes catalyze forward and reverse reactions a set of opposing reactions known as a substrate cycle
What is a phosphatase?
an enzyme that removes phosphate groups from proteins
