biochem exam 4

where does glycolysis happen

cytoplasm

is glycoysis fundamentally aerobic or anaerobic

anaerobic

glycolysis input and output

• input = 1 glucose

• output = 2 pyruvate, 2 net ATP, 2 NADH

phosphofructokinase (pfk) function

• catalyzes phosphorylation of fructose 6 phosphate to fructose 1,6 biphosphate using ATP

• rate limiting step of glycolysis

• exergonic

• irreversible

why is pfk the committing step

• bc before glycolysis could go into glycogen or oher pathways but once it forms fructose 1 6 biphosphate it has to go through glycolysis

master regulator of glycolysis

• fructose 2 6 piphosphate (f26bp)

• activates glycolysis and inhibits gluconeogenesis

• more of it = burn glucose

• less of it = make glucose

wgere does f-2,6 bp come from

made by an enzyme - PFK-2/FBPase -2

• decides whether glycolysis is on or off

hormones and PFK-2/FBPase -2 enzyme

• insulin aka fed state

  • dephosphorylates enzyme so PFK2 is active

    • more F26BP, PFK1, glycolysis

    • after eating = burn glucose

  • glucagon aka fasting

    • phosphorylates enzyme so FBPase2 is active instead

      • lowers everything and increases gluconeogenesis

      • fasting = make glucose

does glycolysis require energy

yes - atp

primary source of atp in cells

mitochondria

• aerobic respiration consumes oxygen to generate atp

at which step of glycolysis does cell begin to produce ATP

• step 7, where phosphate is transferred from 1,3 bisphosphoglycerate to ADP

which correctly describes the fate of dihydroxyacetone phosphate (DHAP) produced in step 4?

• this is the isomer of glyceraldehyde 3 phosphate and it must be isomerized to g3p before continuing in glycolysis

• this is bc the next enzyne is specific to g3p and dhap has a carbonyl in another position

which are the energy investment steps of glycolysis

1-3

step 1 of glycolysis

hexokinase - glucose = glucose 6 phosphate (G6P) using ATP

• traps glucose inside cell

• irreversible

step 2 of glycolysis

phosphoglucose isomerase - G6P = fructose 6 phosphate (F6P) aka the isomer

• aldose to ketose

• prepares molecule for cleavage

• ring opening mechanism and then a closure

step 3 of glycolysis

• phosphofructokinase-1 (PFK1) uses ATP to convert F6P into fructose1,6 biphosphate (F16BP)

  • has 6 carbons

  • committed and rate limiting step of glycolysis

  • irreversible

step 4 of glycolysis

fructose bisphosphate aldolase - cleaves the 6 carbon f16bp to dihydroxyacetone phosphate (DHAP) and glycrealdehyde 3 phosphate (G3P)

• only G3P continues to glycolysis

• lyase clevage reaction

• unfavorable under standard conditions bc pos DG

• reversible reaction

step 5 of glycolysis

triose phosphate isomerase - converts DHAP to G3P so now it can participate in glycolysis

• necessary bc enzyme is specific to g3p and dhap has a carbonyl in wrong place

• reversible step bc pos DG and unfavorable

• kinetically perfect ezyme bc kcat/km approaches maximuum and rate only limited by diffusion

step 6 of glycolysis

glyceraldehyd 3 phosphate dehydrogenase - oxidzes G3P and adds phosphate formin 1-3 bisphosphoglycerate

• nad+ reduced to NADH

• doesnt make ATP but makes the ihgh energy molecule to set up the ATP reaction

step 7 of glycolysis

phosphoglycerate kinase - transfers phosphate from 1-3BPG to ADP forming ATP and 3-phosphoglycerate

• produces 2 atp per glucose

• substrate level phosphorylation - phosphate directly transgerred to ADP

• step where ATP total is break even bc 2 r used and 2 r made

step 8 of glycolysis

phosphoglycerate mutase rearranges phosphate

• 3 phosphoglycerate (low energy) turns into 2 phhosphoglycerate (high energy bc unstable molecule)

• does this bc the next enzyme can remove the waer and form pep and can make more energgy

step 9 of glycolysis

enolase - catalyzes dehydration

• 2 phosphoglycerate = phosphoenolpyruvate (PEP) and water

• high energy phosphate bond - lots of potential energy

step 10 of glycolysis

pyruvate kinase - transfers phoshate from PEP to ADP

• net is 2 pyruvate 2 atp 2 NADH

• forms enol pyruvate which is unstable and then tautomerizes to atp

• second atp payoff step

• inhibitors

  • atp and acetyl coa bc lots of energy

• activated by

  • f16bp and amp = low energy and alr committed to glycolysis

what happens to NADH in anaerobic conditions

• oxidized by lactate or alcohol dehydrogenase and provides more NAD+ for more glycoysis

Which step in glycolysis is most directly affected by arsenate, and what is the mechanism?

• step 6

• this step shoulddd be g3p and pi making bisphosphoglycerate

• arsenate can replace the phosphate (pi) in G3P oxidation step and forms 1-arseno-3-phosphoglycerate

  • veryy unstable and continues w/o giving the phosphate to ADP maeaning no ATP is made soo u make no energy and ur cells #die

thermodynamics and reversibility

a largely negative delta g = exergonic and is usualy ireeversible in cellular conditions

• irreversible reactions act as control points so have the most impact

• target these if you are making a drug

deets about Triose phosphate isomerase

its kinetically perfect?? idk

anaerobic vs aerobic glycolysis end

• aerobic = pyruvate into acetyl coA releasing co2 and enters TCA cycle

• anaerobic = cells regenerate NAD+ so glycolysis can continue

  • humans = pyruvate into lactate (lactic acid fermentation)

  • yeast = pyruvate to ehtanol (alcoholic fermentation)

is going from NAD+ to NADH oxidation or reduction

everytime you add a H its reduction bc it lost an electron

why are coupled reactions in glycolysis important

some reactions are unfavoriable and others are release too much energy so they couple them so that they can be the right amount of favorable aka -DG

is step 1 glycolysis coupled

• yes with hydrolysis so it becomes more favorable

• this also creates a regulatory checkpoint bc it traps glucose inside the cell

• G6P is negative so it cant leave so metabolism can continue

hexokinase operates near what

vmax

hexokinase vs glucokinase

• 1 = muslce and other tissues

  • inhibited by G6P

  • rapid glucose for energy

• glucokinase

  • glucose sensor aka directs excless glucose to glycogen

  • not inhibited by G6P

  • only active when insulin is high

glucokinase (GK) role in pancreatic b cells

1. glucose enters via GLUT2 responding only to high bp

2. gk converts glucose to G6P, giving fuel to glycolysis, TCA, and ETC

3. atp production rises

4. potassium channel closes

5. membrane depolarizes bc no more K+

6. calcium channels open and iy goes in

7. Ca+ triggers insulin vesicles and lowers BGL

alpha cell response to hypoglycemia

• glucagon secretion

1. glucose falls

2. atp/adp ratio drops bc less energy

3. K+ channels close

4. membrane depolarizes

5. calcium opens and triggers glucagon vesicles to highten BGL

why is G6P a branching point

• its fate depends on cellular needs

• can go to glycolysis when it needs atp

• can go to glycogen synthesis when theres a lot of glu

• can go to PPP when biosynthesis or detox is needed

metabolic fate of F6P in diff contexts

• glycolysis

  • committed rate limiting step bc irreversible

• gluconeogenesis

  • reverted back to g6p to make more glucose

• PPP

  • enter via isomerization to g6p and feeds NADPH and ribose 5 phosphate

• hexosamine biosynthethic pathway

  • converted to glucosamine 6 phosphate for glycolysation reactions

PFK 1 activators and inhibitors

• activators aka stimulate glycolysis

  • AMP and ADP

  • fructose 2,6 biphosphate

  • phosphate

• inhibitors aka slow glycolysis

  • atp bc high energy

  • citrate is also high energy

  • H+ (low ph) bc u dont want too much acid

where does F26BP shift the curve toward

• shifts toward hyperbolic aka enzyme is more active when f6p is low

how does phase 2 glycolysis make energy

• oxidation generates NADH

• phosphorylation generate ATP

wdym when everything happens twice in phase 2 glycolysis

• it starts w 2 molecules g3p

• for ex when one nadh is made per g3p its actually 2 nadh total per clucose bc glucose has 2 g3p molecules at this point

How does Fructose-2,6-bisphosphate (F-2,6-BP) affect the kinetics of PFK-1? (idk)

B. It decreases the inhibitory effects of ATP on the enzyme.

F-2,6-BP effectively overcomes the inhibition caused by high ATP

levels, allowing glycolysis to proceed.

substrate level phosphorylation

• phosphate directly trandferred from metabolic intermediate to adp and makes atp immediately

• no oxygen or ETC needde

• happens in cytoplasm

why is 2,3bpg impoirtant for rbc

regulate hemoglobin and releases oxygen to tissues

what are the energy payoff phases of glycolisis

• steps 7 and 10

• 7 = 13BPG - 3PG

• 10 = PEP - pyruvate