Biochem 5 Flashcards
1
Q
carbohydrates
A
- formula Cn(H2O)n
- produced from CO2 and H2O via photosynthesis in plants
- range from as small as glyceraldehyde (Mw=90g/mol) to as large as amylopectin (Mw > 200,000,000 g/mol)
- fulfill a variety of functions, including:
- energy source and energy storage
- structural component of cell walls and exoskeletons
- informational molecules in cell-cell signaling
- can be covalently link with proteins and lipids
2
Q
carbohydrate can be constitutional isomers
A
- an aldose is a carbohydrate with aldehyde functionality (C=O at the end of the molecule)
- a ketose is a carbohydrate with ketone functionality (C=O in the middle of the structure)
- glyceraldehyde- aldotriose
- dihydroxyacetone- a ketotriose
3
Q
important hexose derivatvies
A
- hexose- six carbon sugars
- 5 carbons in the ring and one O
- six carbon comes off as a branch
- hemiketal or hemiacetal
- OH on these sugars tend to get phosphorylated
- one of the carbons can get oxidized all the way from a carbonyl or hydroxyl to a carboxylic acid
- when the carbon it at the end other than the carbonyl carbon it turns the sugar into a uronic acid (important for cartilage)
4
Q
reactivity of carbohydrates: hemiacetals and hemiketals
A
- aldehydes and ketones react with alcohols to form hemiacetals/hemiketals -> they can react with another alcohol to form acetal or ketal
- when aldehydes are attacked by alcohols, hemiacetals form
- when ketones are attacked by alcohols, hemiketals form
- these reactions form the basis of cyclization of sugars
5
Q
hemiacetal and hemiketal formation: glucose and fructose
A
- the formation of hemiacetals is seen when we see sugars cyclize
- cyclization rxn
- glucose- the anomeric C-1 has an aldehyde group on it cyclizes -> forms a 6 member pyranose ring -> beta-D-glucopyranose -> hemiacetal
- pyranose tends to be chair/boat
- fructose- carbonyl carbon is in the C-2 position -> cyclizes -> forms a 5 member furanose ring -> beta-D-fructofuranose -> hemiketal
- reducing disaccharides = accessible hemiacetal
6
Q
6 member ring
A
- tends to form a folded structure
- chair and boat form
- not planar
- ex. half chair being cleaved by lysozyme
7
Q
sugar + hydroxyl group =
A
- two sugars can combine
- first we add methanol onto the carbonyl carbon of the sugars (glucose) -> forms a glucoside
- the bond between the sugar and the methanol is called a glyosidic bond
- methanol can attack in 2 different ways -> alpha and beta orientations
- methyl-alpha-D-glucoside- methanol attacks from bottom
- methyl-beta-D-glucoside- methanol attacks from top
- alpha and beta forms do not have reactive carbonyl carbons anymore -> they are acetal/ketal structures
8
Q
lactose
A
- galactose and glucose form a glycoside bond and bind -> disacchride
- galactose- anomeric carbon in C-1 position is attacked by glucose
- this is a reducing disaccharide -> on the glucose there is still an exposed anomeric carbon hemiacetal structure and chemically that carbonyl carbon can be oxidized very easily
- the hydroxyl of galactose (which is now in the acetal formation with glucose) is not so easily oxidized
- chemical test is employed to detect the presence of hemiacetal/hemiketal -> maltose cannot react with copper but the hemiacetal/hemiketal disaccharide can react with the copper solution
- lactose is a reducing disaccharide with one carbonyl in an accessible hemiacetal structure
9
Q
sucrose
A
- non reducing sugar
- involves the condensation of two sugars via their anomeric glyosidic carbons
- involves the glucopyranose and fructofuranose -> linked via their glyosidic carbons
- no longer an exposed carbonyl in this disaccharide
- two blocked carbonyl carbons (from the linkage)
- it will not react to failing? solution -> no reducing carbons in sucrose
10
Q
polysaccharides
A
- natural carbohydrates are usually found as polymers
- these polysaccharides can be:
- homopolysaccharides (one monomer unit)
- heteropolysaccharides (multiple types of monomer units)
- linear (one type of glycosidic bond)
- branched (multiple types of glycosidic bonds)
- polysaccharides do not have a define molecular weight (all diff sizes)
- this is in contrast to proteins because, unlike proteins, no template is used to make polysaccharides
- polysaccharides are often in a state of flux -> monomer units are easily added and removed as needed by the organism (easily degraded and extended)
- polyglucose- polymer of a simple sugar -> very abundant in potatoes in the form of starch and in the liver in the form of glycogen
11
Q
glycosaminoglycans
A
- linear polymers of repeating disaccharide units
- one monomer is either: N-acetyl-glucosamine or N-acetyl-galactosamine -> (one of the hydroxyls is replaced by an amino group)
- negatively charged:
- uronic acids- (C6 oxidation from an alcohol to a carboxylic acid) -> negative charge
- sulfate esters -> neg charge
- C1 can also be oxidized all the way to carboxylic acid (onic acid)
- this molecules is a polyanion that can bind to water and become extremely hydrated (allows for lubrication)
- extended hydrated molecule:
- minimizes charge repulsion
- forms meshwork with fibrous proteins to form extracellular matrix -> connective tissue and lubrication of joints
- heavily hydrated!
12
Q
common disaccharide units found in glycosaminoglycan
A
- hyaluronate
- dermatan sulfate
- chondroitin-4-sulfate
- chondroitin-6-sulfate
- dermatan sulfate
- keratin sulfate
- heparin
- hyaluronic acid differs from the rest bc it has no sulfate
13
Q
proteoglycans
A
- glycosaminoglycan molecules are linked to core proteins to form proteoglycans
- large
- cartilage
- our tissues have many different core proteins
- linked proteins secure the core protein to the backbone (hyaluronic acid)
- aggrecan is the best studied one -> hyaluronic acid forms the backbone and core proteins branch off -> off each core protein there are a bunch of keratan, dermatan, and chondroitin sulfate oligosaccharides
- principle constituent of cartilage
- sometimes there are other short oligosaccharides that are attached to the core protein (glycosylation)
- bottle brush structure of aggrecan
- capacity to bind large quantities of water
14
Q
gram-positive bacteria
A
- can be stained bc there is no outer membrane
- peptidoglycan cell wall is stained
- peptidoglycan wall is made up of NAG and NAM (cleaved at D site by lysozyme)
- polysaccharides linked by tetrapeptides (which are linked by pentaglycine bridges)
15
Q
gram-negative bacteria
A
-cant be stained due to the outer membrane blocking the peptidoglycan cell wall
16
Q
peptidoglycan
A
- cell wall of bacteria
- alternating co-polymer of N-acetylglucosamine and N-acetylmuramic acid (NAG and NAM)
- off the muramic acid 6 member ring is a lactyl moiety -> has a tetrapeptide attached to it
- the tetrapeptide has some weird amino acids -> D-alanine and glutamic acid is not linked via its alpha carbon but rather its gamma carbon -> isoglutamile linkage
- the tetrapeptide is probably not synthesized the same way proteins are synthesized in mammalian systems
- there are separate enzymes for each attaching each of the amino acids
- another amino acid involved in attaching the tetrapeptide to the alternating co-polymer of NAG and NAM
- the tetrapeptides are linked to each other via a pentaglycine bridge (5 glycine molecules)
- if you compromise the synthesis of any of its components the bacteria may not be killed but it wont be able to replicate
17
Q
penicillin
A
- inhibits the formation of the peptidoglycan specifically by inhibiting the formation of the pentaglycine cross-links
- amide portion of the cyclic structure -> lactam
- lactam linkages are similar to peptide bonds
- lactam ring is subject to attack by enzymes that resemble proteases (beta-lactamases)
- susceptible to cleavage by beta-lactamases
- penicillin does NOT cleave peptidoglycan but just compromises formation
- a good way to kill gram-neg bacteria is to use penicillin bc it blocks formation of pentaglycine bridges (bacteria wont die but it wont proliferate)
- a good way for bacteria to overcome penicillin is to secrete beta-lactamase that inactivates the penicillin
- a good way for a pharmacologist to deal with a beta-lactamase secreting bacteria is to develop a beta-lactamase inhibitor (protease inhibitor)
- penicillin is an good drug if you dont have a beta-lactamase producing bacteria (vice versa)
- penicillin works with beta-lactamase inhibitor though
18
Q
glycoconjugates: glycoprotein
A
- proteins with small oligosaccharides (sugars) attached
- carbohydrate is attached via its anomeric carbon to amino acids on the protein
- common connection occur at Ser, Thr, and Asn
- O-linked glycoproteins and N-linked glycoproteins
- about half of mammalian proteins are glycoproteins
- only some bacteria glycosylate a few of their proteins
- carbohydrates play role in protein-protein recognition
- viral proteins are heavily glycosylated -> this helps evade the immune system
- signal regulator cell growth
19
Q
N-linked glycoproteins
A
- N-linkage- Asn that lies two sugars away from serine or theronine
- Asn is the targeted amino acid thats going to be glycosylated
- turns it into an amino sugar
- the body looks for a consensus sequence of amino acids to find Asn
- consensus sequence- asparagine, C2, carboxyl, always a serine or threonine
- when it sees this sequence Asn is modified
- triggers glycosylation
- occurs in the ER while the protein is being synthesized
- these proteins are co-translationally glycosylated
- separate synthesis of complex oligosaccharides with a lot of mannose residues -> these high content mannose sugars are what is attached to the targeted Asn residue
- once the mannose residues begin to be trimmed back and replaced with other kinds of sugars
- usually longer than their final product when first synthesized and sugars are removed by trimming
- final product: N-linked glycoprotein with a variety of oligosaccharides (no high content of mannose anymore)
- first steps of N-glycosylation involves addition of an oligosaccharide core of 14 sugars
20
Q
O-linked glycoproteins
A
- sugars are put directly on serine and/or threonine one at a time (individual glycosyl transferases carry out)
- sugars are added to hydroxyl of Ser or Thr to form an acetal
- sugars are added sequentially to proteins on the basis of conformational recognition
- made post-translationally
- takes place in the Golgi apparatus
- the entire 3-D structure/conformation of the protein is the recognition mechanism for the glycosylation
21
Q
differences between N- and O- linked glycosylations
A
- N-linked glycosylation sites are recognized by the consensus sequence Ans-X-Ser/Thr -> O-link glycosylation sites are recognized by local conformation of the protein
- N-linked sugars are added to amide nitrogen of Asn (amino sugar) -> O-linked sugars are added to hydroxyl of Ser or Thr to form an acetal
- N-glycosylation takes place as the protein is being synthesized (co-translationally) -> O-glycosylation takes place after the entire protein has been synthesized (post-translationally)
- first steps of N-glycosylation takes place along the ER where the protein is being synthesized -> O-glycosylations take place in Golgi apparatus
- first steps of N-glycosylation involves addition of an oligosaccharide core of 14 sugars -> O-glycosylation involve sequential addition of sugars one at a time, catalyzed by individual glycosyl transferases
22
Q
glycoconjugates: Glycolipids
A
- lipids with covalently bound oligosaccharide
- they are parts of plant and animal cell membranes
- in vertebrates, ganglioside carbohydrate composition determines blood groups
- in gram-negative bacteria, lipopolysaccharides cover the peptidoglycan layer
- lipopolysaccharides are recognized by cell surface proteins known as toll receptors
- lipopolysaccharides are potent signaling molecules referred to as endotoxins
- these lipid side chains can kill you
- causes things like sepsis
- turn off receptors on cells for them or turn of synthesis to deal with these
23
Q
glycoconjugates: membrane proteoglycans
A
- resemble glycosaminoglycans of the proteoglycan components of cartilage
- sulfated glucoseaminoglycans attached to a large rod shaped proteins in cell membrane
- syndecans- contains a single protein that has a single transmembrane domain (spans the membrane) -> hydrophobic
- a bunch of heparan sulfate, chondroitin sulfate, oligosaccharides branch off the syndecan protein that makes it resemble the core protein in cartilage( glycosaminoglycans)
- glypicans- protein is anchored to a lipid membrane
- interact with a variety of receptors from neighboring cells and regulate cell growth
- membrane glycoconjugates have some similarities to proteoglycans, but they are always anchored and signal through their anchors, whereas proteoglycans are generally noncovalently linked to cell, but rather fill the extracellular matrix
24
Q
glycolysis overview
A
- occurs in muscle and brain
- breakdown of glucose in the cytosol
- provide building blocks and ATP
- reversible bc many of the enzymes involved are in equilibrium
- some reaction reverse at the cost of energy too
- probably one of the earliest energy-yielding pathways -> developed before photosynthesis, when the atm was still anaerobic
- the task upon early organisms was how to extract free energy from glucose anaerobically -> 1st: activate it by phosphorylation and 2nd: collect energy from the high energy metabolites
- 10 steps
- glycolytic enzymes catalyze phosphorylation rxns, isomerizations, carbon-carbon bond cleavage, and dehydration
- not oxidative phosphorylation -> substrate level
- ATP is consumed in step 1 and 3 but regenerated in steps 7 and 10 for a net yield of 2 ATP per glucose
- for each glucose, 2 NADH are produced in step 6
25
Q
brief cancer cell overview in metabolism
A
- cancer cells are growing -> want to make more of themselves
- importance of the NADP (oxidizing) step in glycolysis is overshadowed by the branch pathway that is responsible for making NADPH (reducing agent)
- use of different cofactors can control whether were in the energy generating direction or biosynthetic direction (energy consumptive)
26
Q
homolactic fermentation
A
- a means of regeneration NAD
- if you combine glycolysis with homolactic fermentation you can generate ATP at the substrate level without an accumulation of NADH
27
Q
Adenosine triphosphate (ATP)
A
- energy source for biosynthetic rxns
- collection of phosphoanhydride bonds between the gamma and beta and between the beta and alpha linkages
- phosphoester bond- involving ANP
- *most cells dont change their ATP concentration regardless of how active or sluggish they may be metabolically -> what changes a lot are the levels of ADP and ANP (regulatory agents)
- bc ATP may be a goal for degradation but its not a regulatory compound
28
Q
NAD+
A
- nicotinamide adenine dinucleotide
- oxidizing agent in glycolysis
- redox reagent
- mobile 2 e- carrier
- carries 2e- in the form of hydride ion: H+ +2e- -> H-
- usually involved in oxidation of primary alcohol to carbonyl
- R-CH2-OH + NAD+ -> R-CH=O + NADH + H+
- derived from niacin (vitamin B3)
- has an adenine ring, ribose, nicotinamide ring* (oxidized and reduced form)
- phosphorylated to NADP+ -> changes which enzyme uses which redox reagent
- not a high energy intermediate in glycolysis
29
Q
glycolysis: preparatory stage
A
-preparatory to the substrate level phosphorylations (one involving oxidation and the other involving ketoenol totonermization)
30
Q
importance of glucose
A
- excellent fuel
- yields good amount of energy upon oxidation (-2840 kJ/mol glucose)
- can be efficiently stored in the polymeric form
- many organisms and tissues can meet their energy needs on glucose only
- glucose is a versatile biochemical precursor
- many organisms can use glucose to generate:
- all the amino acids
- membrane lipids
- nucleotides in DNA and RNA
- cofactors needed for the metabolism
31
Q
4 major pathways of glucose utilization
A
- glucose is used for biosynthesis and energy (degradation) -> these pathways do double duty- know which ways to activate and inactive for different purposes
- storage:
- can be stored in the polymeric form (starch, glycogen)
- used for later energy needs
- energy production:
- generates energy via oxidation of glucose
- short term energy needs
- production of NADPH and pentoses:
- generates NADPH for use in relieving oxidative stress and synthesizing fatty acids
- generates pentose phosphates for use in the DNA/RNA biosynthesis
- structural carbohydrate production:
- used for generation of alternate carbohydrates used in cell walls of bacteria, fungi, and plants
32
Q
NADPH
A
-used for biosynthetic rxns
33
Q
NAD+
A
-oxidation of glucose
34
Q
some products of glucose
A
- extracellular matrix and cell wall polysaccharides (synthesis of structural polymers)
- glycogen, starch, sucrose (storage)
- ribose-5-phosphate (oxidation via pentose phosphate pathway
- pyruvate (oxidation via glycolysis)
35
Q
polymerization
A
- way of avoiding excess increase in osmotic pressure in a cell
- if we stored glucose as glucose the cytosolic environment would be hypertonic
- when we polymerize into glycogen -> its fine
- versatile
- means we need a way to break down to get glucose
36
Q
importance of glycolysis
A
- sequence of enzyme-catalyzed rxns by which glucose is converted into pyruvate
- pyruvate can be further aerobically oxidized
- pyruvate can be used as a precursor in biosynthesis
- some of the free energy is captured by the synthesis of ATP and NADH
- important for energy and equally important for biosynthesis (especially neoplastic rapidly dividing cells)
- research of glycolysis played a large role in the development of modern biochemistry:
- understanding the role of coenzymes
- discovery of the pivotal role of ATP
- develop of methods for enzyme purification
- inspiration for the next generations of biochemists
37
Q
Glycolysis prep stage: Step 1: Hexokinase
A
- phosphorylation of C6 of glucose
- hexose rxn -> clever way of trapping glucose inside cells
- hexokinase- first priming rxn
- kinases- enzymes that transfer Pi from ATP to a substrate
- hexokinase also phosphorylates mannose and fructose
- glucose + ATP -> glucose-6-phosphate (G6P) + ADP + H+
- stays in the cytosol
- its much easier to put the P onto the C6 than to C1 -> makes a phosphoester
- harder to put on C1 bc its apart of a hemiacetal
- example of a coupled rxn
- this rxn is energy consuming -> ATP is the phosphate donor and the energy is used to put the phosphate on C6
38
Q
glucose-6-phosphate (G6P): issues with phosphatases
A
- product of step one of glycolysis
- there are a lot of phosphatases inside cells
- its always a risk making phosphorylated compounds bc a phosphatase can come around and take the phosphate off
- the trick to avoid this is to: keep water away
- hydrophobic interior of the enzyme helps
- ATP and glucose are bound in a deep cleft that excludes water -> eliminates risk of hydrolysis of the phosphate group off
- glucose binding causes a large conformational shift, excluding water and bring the 6’-OH close to the ATP
- this is not unique to hexokinase (common strat)
39
Q
hexokinase vs. glucokinase
A
- most cells have hexokinase that has a high affinity (low Km) for glucose -> easy for glucose to be saturating the enzyme within the interior of the cell
- liver is self-less -> not worried about acquiring glucose for its own needs
- in the liver glucokinase is a regulator of blood glucose
- the regulator glucose-6-phosphatase undoes the selective protection of the phosphate in the liver (untraps)
- glucose-6-phosphatase takes the phosphate off and glucokinase puts the phosphate on -> rate of phosphate transfer to glucose by glucokinase is dependent on the concentration of glucose
- phosphorylation of glucose is trapping it in the liver (low blood glucose) and de-phosphorylation is releasing it (high blood glucose)
- the Km for glucokinase is close to the normal physiological levels of glucose
- hypoglycemia (low blood glucose) -> rate of phosphorylation of glucose decreases
- hydrolysis of glucose-6-phosphate by glucose-6-phosphatase rapidly maintains and makes sure that free glucose is released by the liver during hypoglycemia
- during hyperglycemia the rate at which glucokinase phosphorylates glucose increases -> lowers the blood glucose levels
- hexose is inhibited by the product (G6P) of its rxn where glucoinase isnt
40
Q
glucose-6-phosphate: different uses
A
- in the liver we can break it down back to glucose (liver stores glucose as glycogen)
- can become acetyl-CoA (2 carbons) -> this is the product of pyruvate (made in glycolysis)
- acetyl-CoA is a precursor for fatty acids, phospholipids, cholesterol
41
Q
glycolysis prep stage: step 2: phosphoglucose isomerase (PGI)
A
- glucose-6-phosphate is converted to fructose 6-phosphate (F6P)
- converts C1-OH to a simple hydroxyl (not a hemiacetal)
- *now it will be easy to phosphorylate C1 of fructose-6-phosphate
- catalyzes reversible isomerization using acid-base catalysis with an enediol intermediate
- phosphoglucose isomerase facilitates this conversion
- important intermediate: enediol
- enediol is a good way to move a single carbonyl group
- converts the 6 membered aldose (glucose-6-phosphate) to the 6 member ketose (fructose-6-phosphate)
- equilibrium rxn -> no regulation
- enediol mechanism
42
Q
glycolysis prep stage: step 3: phosphofructokinase (PFK1)
A
- committed step -> key to regulatory site
- second priming rxn
- coupled rxn
- irreversible in cells
- biphosphate: 2 phosphates not linked to one another
- fructose-6-phosphate + ATP -> fructose-1,6-biphosphate (FBP) + ADP + H+
- phosphate is now on C1 and C6
- often not at equilibrium
- PFK1- consumes ATP
43
Q
glycolysis: committed step regulation
A
- PFK is regulated
- PFK- a dimer, two binding sites, two allosteric type binding sites on each subunit (4)
- similar to NWC model of hemoglobin
- binding of negative allosteric effectors results in decrease in affinity of PFK for its substrate F6P (fructose-6-phosphate)
- PFK is negatively regulated by binding of ATP in the allosteric site (ATP in the normal binding site facilitates phosphorylation of F6P)
- K type allosteric enzyme- what changes is not the catalytic constant -> rather the affinity
- recall: ATP concentration is relatively constant…
- under normal conditions PFK is negatively regulation by the constant high levels of ATP
- if there is a lot of metabolic activity some ATP will be converted to ADP and ANP
- ANP is a positive allosteric effector for PFK -> bound with greater affinity than ATP
- ANP turns the affinity of PFK for F6P up -> F6P is bound with greater affinity when ANP levels rise
- if glycolysis is a way of making ATP the cell knows its going to need more ATP when it encounter high levels of ANP (this means cell is using a lot of ATP) -> this allows the cell to make more ATP by binding to F6P and committing to glycolysis
44
Q
fructose
A
- in the liver fructose is converted by fructokinase to fructose-1-phosphate
- fructose-1-phosphate is not a substrate for PFK
- it is a substrate for the next enzyme (aldolase)
- if fructose-1-phosphate can be cleaved by fructose aldolase -> you carryout glycolysis and bypass PFK in the liver
- PFK is the regulatory step in glycolysis
- if you feed the liver fructose-1-phosphate it can carry out glycolysis in an unregulated way
- the more fructose you eat the more fructose-1-phosphate you make and the more glycolysis -> glyceraldehyde-biphosphate and dihydroxyacetone is a byproduct of this and get reduced and converted to glycerol phosphate which is used to drive the synthesis of fats -> fatty liver
45
Q
glycolysis prep stage: step 4: aldolase
A
- aldose converts fructose-1,6-biphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP)
- reversible aldol condensation
- always in equilibrium
- efficient
- cleavage rxn
- change in numbering in this step!
- in the liver aldolase cleaves fructose-1-phosphate (bypasses regulatory step of glycolysis)
46
Q
mechanism of Class 1 aldolase
A
- aldolase makes use of a schiff base and an aldol condensation
- schiffs base formation
47
Q
glycolysis prep stage: step 5: triose phosphate isomerase (TIM) enediol intermediate, like PGI
A
- use enediol mechanism to convert the hydroxyacetone phosphate to glyceraldehyde-3-phosphate (an aldose)
- moves a carbonyl from the keto position to the aldol position
- glyceraldehyde-3-phosphate and dihydroxyacetone phosphate are both trioses
- DHAP is the ketose
- G3P is the aldose
48
Q
glycolysis payoff stage: step 6: GAP dehydrogenase
A
- glyceraldehyde-3-phosphate (GAP) is converted to 1,3-biphosphoglycerate (1,3-BPG) via glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
- 1,3-biphosphoglycerate is a mixed anhydride -> high energy phosphate
- dehydrogenases- involved in oxidation/reduction reactions
- couples favorable oxidation to unfavorable phosphorylation through covalent intermediate thioester
- dihydroxyacetone phosphate (from last step) can easily be converted to glycerol phosphate -> important for adipocyte where it esterifies with fatty acids to make neutral fats
49
Q
GAP dehydrogenase mechanism: step 6
A
- covalent intermediate (several)
- the enzyme has prepositions sulfhydryl group and NAD+ in the pocket (non-covalently bound)
- the substrate binding portrays classic michaelis menten behavior
- reaction of the carbonyl of gylceraldehyde-3-phosphate with a thiol on the enzyme -> forms a thiohemiacetal (covalently bound)
- thiohemiacetal is oxidized by an NAD+ that is bound to the enzyme
- NAD+ is reduced to NADH and thiohemiacetal is oxidized to a thioester (high energy)
- NADH is loosely bound
- phosphorolysis- inorganic phosphate cleaves the thioester forming a mixed anhydride (high energy) between phosphate and the carboxyl of glycerate acid
- this conserves energy of the thioester (high energy intermediate) in acyl-phosphate
50
Q
glycolysis payoff stage: step 7: phosphoglycerate kinase
A
- capturing the high energy bond that is in a mixed anhydride by reacting it with ADP
- use ADP to cleave the mixed anhydride of 1,3-BPG
- mixed anhydride phosphorylates ADP
- products are ATP and 3-phosphoglyerate (3PG)
- substrate level phosphorylation
- 1,3-biphosphoglycerate (1,3-BPG) is converted to 3-phosphoglycerate (3PG) via phosphoglycerate kinase (PGK)
- highly reversible when coupled (bc were converting a high energy phosphate (mixed anhydride) to another high energy phosphate)
51
Q
phosphoglycerate kinase (PGK)
A
- resembles hexokinase
- hides phosphates inside hydrophobic pocket
- this is so the 1 phosphate on 1,3-biphosphoglyceric acid is not immediately hydrolyzed by water
- PGK catalyzes a coupled reaction
- source of ATP at the substrate level
52
Q
glycolysis payoff stage: step 8: phosphoglycerate mutase rxn
A
- mutase- subclass of isomerases, moves functional group from one position to another on substrate
- moves the 3-phosphate from the 3PG mixed anhydride to the 2 position on glyceric acid
- when its on the 1 position its a mixed anhydride when its on the 2 position its not so impressive in energy
- mutase does this by making distinct intermediates -> uses enzyme bound 2,3-BPG equal in concentration to the enzyme itself (catalytic quantities
- phosphohistidine is used -> requires an ATP to make this sometimes
- substrate binds ->2,3-BPG is made using phosphohistidine which phosphorylates the 2nd position -> free histidine is present -> 2,3-BPG re-phosphorylates the histidine with the phosphate from the 3rd position -> forms 2PG-phosphoenzyme
53
Q
hemoglobin
A
- cannot work with catalytic (micromolar) quantities of 2,3-DPG -> needs millimolar
- Rapoport Luebering shunt
- the product of GAPDH is 1,3-BPG
- in RBC there is a DPG phosphotase and DPG mutase
- DPG mutase- is to take 1,3-DPG and convert it to 2,3-DPG
- the enzyme becomes more and more active as the pH goes down
- lowering the pH is a call to release O2 to the tissues -> elevate 2,3-DPG -> deoxygenation (negative allosteric effector)
- can make as much as needed
- 2,3-DPG phosphotase- converts 2,3-DPG to 3-phosphoglycerate -> sacrifices an ATP to make this (recall that PGK can make 3-phosphoglycerate without using ATP)
- bypasses the chance to make ATP in order to make substrate level 2,3-DPG
- wastes an ATP in a cell that doesnt need a lot of ATP
54
Q
glycolytic intermediates: O2 affinity
A
- people who lack hexokinase enzyme -> make lower levels of all the glycolytic intermediates
- includes 1,3-DPG making it harder to make 2,3-DPG -> left shifted O2 affinity curve (high affinity)
- people who lack the last enzyme in the glycolytic pathway (pyruvate kinase) -> build up glycolytic intermediates
- high levels of 2,3-DPG -> right shift -> low affinity for O2
55
Q
glycolysis payoff stage: step 9: dehydration/hydration
A
- 2-phosphoglycerate (2PG) gets dehydrated via the enzyme enolase -> forms phosphoenolpyruvate (PEP)
- enolase-metal dependent enzyme; converts low energy to high energy phosphate
- enolphosphate (highest energy phosphate)
- inhibitor for enolase is fluoride -> fluoride prevents the binding of the metal (Mg) that is needed for the rxn
- reversible
56
Q
glycolysis payoff stage: step 10: pyruvate kinase
A
- substrate level phosphorylation
- capture of the energy in the enolphosphate using ADP
- similar to PGK rxn
- phosphoenolpyruvate (PEP) is converted to pyruvate and ATP via pyruvate kinase (PK)
- largest free energy drop in glycolysis (-61.9kJ/mol) pulls entire pathway forward by mass action
- not reversible
- first product of rxn between ADP and phosphoenol-pyruvate (PEP) is the enol form of pyruvic acid -> highly reversible; however, it immediately tautomerizes into the keto form
- keto enol tautomerization drives the overall rxn and prevents the rxn from reversing (coupled) and permits the production of ATP using high energy from PEP
- pyruvate kinase is a ATP source at the substrate level
57
Q
products of glycolysis
A
- 1 ATP made at phosphoglycerate kinase
- another ATP made at pyruvate kinase
- one NADH made at GAPDH
- if we dont use the NADH there will be problems bc we need the NAD+ for the next glycolysis cycle -> no glycolysis
58
Q
oxidation of GAP by glyceraldehyde-3-phosphate dehydrogenase
A
- generation of a high energy phosphate compound incorporates inorganic phosphate -> allows for net production of ATP via glycolysis
- first energy yielding step in glycolysis
- oxidation of aldehyde with NAD+ gives NADH
- active site cysteine forms high energy thioester intermediate
- subject to inactivation by oxidative stress
- thermodynamically unfavorable/reversible -> coupled to next reaction to pull forward
59
Q
1st production of ATP by phosphoglycerate kinase
A
- substrate level phosphorylation to make ATP
- 1,3-biphosphoglycerate is a high energy compound -> can donate the phosphate group to ADP to make ATP
- kinases are enzymes that transfer phosphate groups between ATP and various substrates
- highly thermodynamically favorable/reversible -> is reversible bc of coupling to GAPDH rxn
60
Q
migration of the phosphate by phosphoglycerate mutase
A
- be able to form high energy phosphate compound
- mutases catalyze the (apparent) migration of functional groups
- one of the active-site histidines is posttransitionally modified to phosphohistidine
- phosphohistidine donates its phosphate to 3-phosphoglycerate at the 2-carbon oxygen before retrieving another phosphate from the 3-carbon oxygen
- note that the phosphate from the substrate ends up bound to the enzyme at the end of the rxn
- thermodynamically unfavorable/reversible -> reactant concentration kept high by PGK to push forward
61
Q
dehydration of 2-PG to PEP
A
- generate a high energy phosphate compound
- 2-phosphoglycerate is not a good enough phosphate donor to generate ATP
- 2 negative charges in 2-PG are fairly close
- but loss of phosphate from 2-PG would give a secondary alcohol with no further stabilization
- slightly thermodynamically unfavorable/reversible -> product concentraton kept low to pull forward
62
Q
2nd production of ATP by pyruvate kinase
A
- substrate level-phosphorylation to make ATP
- net production of up to 2 ATP/glucose (1 ATP per GAP) depending on diversion of DHAP to fat synthesis
- loss of phosphate from PEP yields an enol that tautomerizes into ketone
- tautomerization effectively lowers the concentration of the rxn product -> drives the rxn towards ATP formation
- pyruvate kinase requires divalent metals (Mg++ or Mn++) for activity
- highly thermodynamic favorable/irreversible -> regulated by ATP, divalent metals, and other metabolites
63
Q
homolactic fermentation
A
- key to keeping the glycolytic pathway going
- in RBC that have no mitochondria and muscles
- take pyruvate and reduce it to lactic acid
- regenerate the NAD+ to keep the NADH running for glycolysis
- NAD is made for GAPDH
- lactate is the product of anaerobic glycolysis
- if there is no O2 to do anything with NADH -> convert pyruvate to lactate
- generates a build up of organic acids (lactic acid) -> 2nd best source of protons
- in yeast it converts pyruvate to ethanol
- lactate dehydrogenase- enzyme that carries this out (highly reversible)
- lactate dehydrogenase uses NADH to convert pyruvate to lactate and NAD+
- if there is mitochondria it uses the krebs cycle to convert NAD+ to NADH
- this step is not regulated
64
Q
yeast
A
- oxidize pyruvate to acetaldehyde (compound that reduces a hangover by suppressing ethanol)
- decarboxylation of a pyruvate (alpha ketoacid) to acetaldehyde
- trick pyruvate to think its like beta keto acid (decarboxylates spontaneously)
- conversion to pyruvate to acetaldehyde is dependent on a co-factor -> thiamine pyrophosphate (TPP)
- the C=N in TPP is identical to the one found in cyanide
- reduction of acetaldehyde to ethanol via alcohol dehydrogenase (highly reversible) -> carbonyl is reduced to a hydroxyl
- alcohol dehydrogenase uses and converts NADH to NAD+
65
Q
cyanide
A
- benzaldehyde and shake with cyanide -> 2 carbonyls of benzaldehyde react directly and combine in the presence of cyanide -> form benzaline
- condensation product
- cyanide reacts with one of the carbonyls -> the product is a cyanohydryl
- forms a double bond
- carbonyl is moved one carbon away -> now you can carry an aldehol condensation with another carbonyl
- Thiamine pyrophosphate is the biological form of cyanide that permits the carbonyls to react directly -> product is a decarboxylation of pyruvate to form acetaldehyde
