BIOL0280 - Biochem Module 2 - Integration of Metabolic Pathways

What process generated the oxygen in our atmosphere?

Oxygenic phosphorylation

What is the area inside the inner chloroplast membrane but outside of the thylakoids? What is a stack of thylakoids called?

Stroma, granum

How many layers of membranes does a chloroplast have?

3

What are the two productive outcomes of an increase in electron potential energy resulting from photon absorption?

Energy transfer to nearby atom (“exciton” transfer), electron easier to transfer to nearby atom (oxidation)

What is the physical structure of chlorophyll?

Magnesium containing porphyrin ring with conjugated alkenes and a long hydrocarbon tail (hydrophobic)

What is the function of accessory pigments in chloroplasts?

Extend range of light absorption

Where are light harvesting complexes found and what are they comprised of?

Thylakoid membrane, antenna complex (many pigment molecules) + reaction center (special pair)

Is the ratio of light harvesting complexes to reaction centers 1:1?

No

What is the relationship between the light dependent and light independent reactions in chloroplasts?

Light-dependent reactions use H2O to reduce NADP+ and synthesize ATP, generating O2 as a byproduct, and the light-independent reactions reduce NADPH and hydrolyze ATP to turn CO2 into triose phosphates

What is the oxygen evolving complex (OEC)?

The part of photosystem II located on the thylakoid lumen side which oxidizes 2H2O to O2 and 4H+ via a series of photochemical redoxes with Mn

Where is the Calvin Cycle located?

Chloroplast stroma

Where do the excited electrons of the chlorophyll special pair go in photosystem II and how are they replaced?

PQ inside the thylakoid membrane, OEC water oxidation from lumen

How is plastohydroquinone oxidized and where do its electrons go? What is this analogous to in the ETC?

Two step oxidation by cytochrome b6f complex, UQH2 electron transfer via the Q cycle to Cyt-C by complex 3

Where is plastocyanin found and and how many electrons can it carry?

Thylakoid lumen, 1

What is the proton removal/pumping behavior of cytochrome b6f and from where to where?

remove 2H+ (during second PQH2 oxidation), pump 4H+ (two during each PQH2 oxidation), from stroma to lumen

Where does one excited electron of the chlorophyll special pair go in photosystem I and how is it replaced?

Ferredoxin (Fd) in stroma, plastocyanin from lumen

Where does ferredoxin shuttle its electron to produce NADPH versus ATP? What are each of these called?

NADPH reductase (non-cyclic electron transport), Cyt-b6f (cyclic electron transport)

Which electrons are higher energy/redox potential, electrons leaving photosystem 1 or 2?

PS1

Where are the CFo and CF1 subunits of ATP synthase located in chloroplasts? How many protons are required to cause one full rotation of the CFo subunit?

Thylakoid membrane, stroma, 12

What does the Calvin Cycle require in terms of ATP/NADPH input from the light-dependent reactions to fix 3 CO2?

9 ATP, 6 NADPH

What is photophosphorylation?

ATP synthesis driven by a light-generated proton motive force

What is the structure and composition of appressed thylakoids? What about non-appressed?

Tightly packed (minimal access to stroma) + ^^PSII and LHC (optimizes light capture), unstacked (^access to stroma = Fd/NADP+/ADP+ access) + ^^PSI and ATP synthase

What structure mediates the connection between adjacent thylakoid membranes in a granum? How is thylakoid stacking changed to convert between the appressed and non-appressed states?

LHC, phosphorylation/hydrolysis of Thr residues in LHC

What is the state of LHC in appressed thylakoids and what enzyme converts it to non-appressed?

Normal Thr from LHC embedded in adjacent membrane, protein kinase

What is the state of LHC in non-appressed thylakoids and what enzyme converts it to appressed?

Phosphorylated Thr on LHC free in stroma, protein phosphatase

What are the three stages of the Calvin Cycle?

CO2 fixation, 3-PG reduction to G3P, regeneration of acceptor (ribulose-1,5-BP)

What does RuBisCO stand for? What are some of its properties?

Ribulose-1,5-BP carboxylase/oxygenase, very slow + LARGE amounts needed in chloroplasts, catalyzes unwanted side reactions if O2 binds instead of CO2, Mg2+ chelates in active site

What is the activated intermediate form of R-1,5-BP in the first stage of the Calvin Cycle and why is it necessary to form it?

Enediolate, CO2 is a very poor electrophile

How many of the G3P generated in the second phase of the Calvin Cycle are necessary to regenerate how many R-1,5-BP?

5/6, 3, 3

What is the balanced equation of the second stage of the Calvin cycle?

6 3-PG + 6 ATP + 6 NADPH + 6 H+ = 6 G3P + 6 ADP + 6 Pi + 6 NADP+

Where does the one extra G3P generated in the Calvin Cycle go?

Glycolysis or starch/sugar synthesis

What are the two steps of stage 2 of the Calvin Cycle?

3-PG phosphorylation via ATP hydrolysis then 1,3-BPG reduction via redox with NADPH

How is G3P, a triose, converted to R-1,5-BP, a pentose, in the third stage of the Calvin Cycle? How much ATP does this require?

Interconversion of G3P and DHAP, 3 total, 1 for each pair of G3P/DHAP

How is the Calvin Cycle indirectly activated by light?

^stromal Mg2+ conc. + ^stromal pH = ^RuBisCO activity (over 100-fold)

What is the glucose usage/glycogen storage/GNG capabilities of the brain?

^^glucose usage, no glycogen storage, no GNG (relies on blood sugar)

What is the glycogen storage/GNG capabilities of the liver?

^^glycogen storage + GNG (90% for body), maintains blood sugar

What are the GNG capabilities of the kidney?

10% of GNG for body, mostly during fasting

What is the glucose usage/glycogen storage/GNG capabilities of skeletal muscle?

^burn glucose for ATP, stores glycogen but won’t export glucose, export lactate under anaerobic (goes to liver for GNG)

What is the Cori Cycle?

Lactate produced by skeletal muscles during anaerobic respiration is exported to the liver to undergo GNG and return to skeletal muscle as glucose

What is the name for the process which stores glucose as glycogen? Where does it occur?

Glycogenesis, mostly muscles but also liver long term

What are the structural features of glucose storage as glycogen in the body?

Glycogenin protein core, mostly alpha1>4 glucose with alpha 1>6 branches every 8-12

What are the advantages of glycogen’s structure?

Reduce need for water to solubilize, 3D structure allows for parallel processing

At what position is the UDP attached to glucose in the second step of glycogenesis? What is the utility of this?

1, ^reactivity, ^binding energy w/ enzymes, separate pool of glucose set aside for glycogenesis

What must happen before UDPG is formed in glycogenesis?

Isomerization of G-6-P to G-1-P

What are the two general steps for synthesizing sugar nucleotides? (N = identity of nucleotide, A/T/C/G/U) What is the driving force of this reaction? Is it reversible?

Sugar phosphate Sn2 at inner phosphoric anhydride via NDP-sugar phosphorylase, pyrophosphate (PPi) split into x2 Pi via inorganic pyrophosphatase, second step, no

How many of the first molecules of glucose does glycogenin add to form the primer strand of glycogen? How? What end does it remain covalently bound to?

7, ATK by terminal Tyr, reducing end

What enzymes catalyzes the formation of the alpha1>4 linkages in glycogenesis? What about the alpha1>6?

Glycogen synthase, glycogen branching enzyme

What kind of end is the free end of a glycogen chain?

Non-reducing

How does glycogen branching enzyme catalyze the formation of the alpha1>6 linkages in glycogen?

Catalyzes transfer of 6-7 residue terminal fragment from non-reducing end of the branch point to the C6 hydroxyl (on same or different chain)

What is the name of the process which degrades glycogen?

Phosphorolysis

What is the advantage of phosphorolysis in glycogen breakdown over hydrolysis?

Preserve bond energy

Are the enzymes for glycogenesis and phosphorolysis of glycogen the same?

No (synthase v. phosphorylase, branching v. debranching)

What is the phosphorolysis activity of glycogen phosphorylase?

Sequentially converts terminal residues to G-1-P but stops 4 before branch

What is the phosphorolysis activity of glycogen debranching enzyme?

Transferase activity shifts 3 residues to non-reducing end of same or different strain, glucosidase activity resolves alpha1>6 via hydrolysis

What are the a and b forms of glycogen phosphorylase? What are they reciprocally regulated with?

Active/baseline, glycogen synthase a/b

What effect does high levels of epinephrine and glucagon have on glycogen synthase/phosphorylase?

Deactivate/activate

What steps of glycolysis need to be bypassed in GNG due to their irreversibility? What are the features of these bypass reactions?

1, 3, 10, effectively irreversible, reciprocally regulated

Where does oxaloacetate for the first bypass of GNG come from? Why can’t it come from L-malate oxidation in the TCA cycle?

Pyruvate carboxylase in matrix, substrate channeling with citrate synthase

What is the pathway of the first bypass of GNG if starting with lactate?

Lactate and NAD+ = pyruvate and NADH via lactate dehydrogenase, pyruvate > matrix, pyruvate + bicarb = oxaloacetate via pyruvate carboxylase, then = PEP by mitochondrial PEP carboxykinase, then > cytosol

What is the pathway of the first bypass of GNG if starting with pyruvate?

Pyruvate > matrix, pyruvate + bicarb = oxaloacetate via pyruvate carboxylase, then NADH and oxaloacetate redox > L-malate and NAD+ via mitochondrial malate dehydrogenase, exported to the cytosol, malate and NAD+ = oxaloacetate and NADH via cytosolic malate dehydrogenase, then = PEP via cytosolic PEP carboxykinase

Why is GNG starting with lactate able to follow a more efficient pathway during the first bypass?

Already able to generate cytosolic NADH which is required for GNG

How is bicarbonate activated in order to transfer to the swinging arm of pyruvate carboxylase as CO2? What is the swinging arm cofactor called?

ATP hydrolysis, biolectin

How is PEP formed from oxaloacetate by the carboxykinase?

Decarboxylation and GTP hydrolysis

How many ATP equivalents are required for the first bypass of GNG?

2

What is pyruvate carboxylase reciprocally regulated with? What molecule does the regulating?

PDH complex, Acetyl CoA (activate carboxylase, deactivate dehydrogenase)

What is PFK-1 reciprocally regulated with? What molecule does the regulating?

FBPase-1, F2,6BP (glucose sensor = activates glycolysis, deactivates GNG)

What does FBPase-1 catalyze?

Hydrolysis of F-1,6-BP to F-6-P (no phosphoryl transfer, H2O in and free Pi out)

What enzyme(s) regulate the cellular levels of F-2,6-BP? What is the result of it being phosphorylated?

Dual function PFK-2 and F-2,6-BPase, increase Km for F-6-P = ^F-2,6-BPase activity

What molecule activates the PFK-2 subunit and inactivates the FBPase-2 subunit? What is it indicative of? What does it result in?

Insulin, high blood sugar, high F-2,6-BP

What molecule inactivates the PFK-2 subunit and activates the FBPase-2 subunit? What is it indicative of? What does it result in?

Glucagon, low blood sugar, low F-2,6-BP

What enzyme catalyzes the third bypass of GNG and how is it regulated?

Glucose-6-phosphatase, compartmentalization (only expressed on lumen side of ER) and expression (only expressed in tissues which export glucose, liver/kidney)

How many total ATP equivalents and NADH are used in GNG?

6, 2

Why do cells maintain high [NAD+] v. high [NADPH]

Drive oxidation of glucose, drive anabolic

What are the two phases of the pentose phosphate pathway?

Oxidative and non-oxidative

What is overall accomplished in the oxidative phase of PPP?

G-6-P oxidation to Ribose-5-phosphate, NADP+ reduction x2

What do isomerases/epimerases do in the PPP non-oxidative phase? What about transaldolases/ketolases? Do they alter the total number of carbons in the backbones?

Alter phosphopentose backbone, move portions of carbohydrate backbone between molecules, no

How would a cell that needs ribose AND NADH run the PPP? What kind of cell scenario is this?

Only phase 1 (stop at R-5-P to build DNA), rapidly dividing cells

How would a cell that only needs ribose run the PPP? What kind of cell scenario is this?

Reverse phase 2 (make ribose from G3P and F6P w/o making NADPH), pre-mitosis cells

How would a cell that only needs NADPH run the PPP? What kind of cell scenario is this?

Run phase 1 and phase 2 (make NADPH and turn R-5-P into G6P to run again until it’s all gone), RBC or adipose tissue

How would a cell that needs NADPH and ATP run the PPP? What kind of cell scenario is this?

Run phase 1 and phase 2 (make NADPH and turn R-5-P into G3P/F6P to run glycolysis), cells under oxidative stress

Why are lipids (fatty acids) more efficient for storing energy than carbohydrates?

^reduced carbons, hydrophobic = don’t need water to solubilize

What protein solubilizes fatty acids and transports them in the blood?

Albumin

What solubilizes fatty acids in the gut? How?

Amphipathic bile salts, form mixed micelles

What are the four types of lipoproteins?

Chylomicrons, VLDLs, LDLs, HDLs

What are the features/functions of chylomicrons?

Transport exogenous lipids from intestines > body, in blood only after meal

What are the features/functions of VLDLs?

Produced by liver, contains endogenous triglycerides and cholesterol, deliver triglycerides to peripheral tissue

What are the features/functions of LDLs?

VLDLs after remove triglycerides, deliver endogenous cholesterol to peripheral tissue

What are the features/functions of HDLs?

Deliver excess cholesterol/wandering LDLs from peripheral tissues back to liver

What hydrolyzes glycerolipids during lipid metabolism? Where do the glycerol and free FA go respectively?

Lipases, converted to DHAP for glycolysis in cytosol, diffuse/shuttled into mitochondrial matrix

What is the amount of carbons in a free fatty acid at and above which it is considered ‘long’ (needs to be shuttled into the matrix)?

14

Where are short and long chain FA activated respectively? How many ATP equivalents does this require and why?

Matrix, cytosol, 2 (ATP > AMP in one molecule)

What is the two step process of FA activation?

Adenylation of carboxylate end = fatty acyl-adenylate + PPi, PPi immediately hydrolyzed to 2 Pi, CoA-SH attacks carbonyl carbon and diplaces AMP, generating fatty acyl-CoA thioester

How are long chain FA transported in the mitochondrial matrix after being activated?

Temporarily replace CoA with carnitine in cytosol, regenerate thioester once in matrix

What is the name for the process which oxidizes free FA in the matrix and what does it accomplish each round?

Beta oxidation, remove 2C at a time as Acetyl-CoA and produce 1 NADH + 1 FADH2

What process do the first three steps of beta oxidation mimic exactly?

Final three steps of TCA cycle

What are in the inputs/outputs of each of the four steps of beta oxidation?

Thioester hydrogenation to alpha-beta unsat. thioester + FADH2, add water to hydrogenate beta to thioester, oxidize new -OH to ketone + NADH, thiolase cleaves to release Acetyl-CoA + add CoA-SH to form new thioester

What are the enzymes and intermediates of beta oxidation?

acyl-CoA + acyl-CoA dehydrogenase = trans-delta^2-enoyl-CoA, + enoyl-CoA hydratase = L-beta-hydroxy acyl-CoA, + beta-hydroxy acyl-CoA dehydrogenase = beta-keto acyl-CoA, + acyl-CoA acetyltransferase (thiolase) = acyl-CoA + Acetyl-CoA

How is FADH2 produced by beta oxidation used by the electron transport chain?

ETF shuttles FADH2 from Acyl-Coa dehydrogenase to membrane-bound ETF:Q oxidoreductase to feed into QH2 pool

How are saturated FA catabolized during beta oxidation?

Convert from cis to to trans-enoyl CoA

How are free FA with an odd number of carbons catabolized during beta oxidation?

Final product is 3C propionyl-CoA, convert to succinyl-CoA via methylmalonyl-CoA for use in TCA cycle (anaplerotic!)