biochem glycolysis

T or F: rxns in glycolysis often go backwards

F: reactions do not go backward unless there’s a large backup of an intermediate (so it is rare for them to go backwards because intermediates are easily consumed)

Glycolysis Preparatory phase

Step 1: Glucose to Glucose 6 Phosphate

Step 2: Glucose 6 phosphate to Fructose 6 phosphate

Step 3: Fructose 6 phosphate to fructose 1,6 bisphosphate

Step 4: fructose 1,6 bisphosphate to GAP and DHAP

Priming steps of glycolysis

• steps that use ATP

• step 1 (phosphorylation of glucose)

• step 3 (phosphorylation of fructose 6 phosphate)

Step 1 of Glycolysis

• glucose to glucose 6 phosphate (phosphorylation)

• IRREVERSIBLE RXN

• hexokinase

• first ATP molecule used

Step 2 of glycolysis

• glucose 6 phosphate to fructose 6 phosphate

• phosphohexose isomerase

step 3 of glycolysis

• fructose 6 phosphate to fructose 1,6 bis phosphate (another phosphate added)

• IRREVERSIBLE RXN and most regulated step

• phosphofructokinase-1 (PFK-1)

• uses second ATP

step 4 of glycolysis

• fructose 1,6 bis phosphate to GAP and DHAP

• aldolase

• six carbon is split into two 3 carbon pieces (GAP and DHAP)

step 5 of glycolysis

• DHAP to GAP

• triose phosphate isomerase

• catalyzes interconversion between DHAP and GAP (pushes toward GAP so there is two GAP molecules)

• GAP has an aldehyde on the end (glyceraldehyde 3 phosphate)

hexokinase vs glucokinase

• hexokinase

  • works in the muscles mostly

• glucokinase

  • works in the liver mostly

  • exists in the nucleus and is onlly released when there’s a low concentration of glucose in the cell

    • glucokinase regulatory protein binds to enzyme and traps it in the nucleus so glucose is not phosphorylated in the cytoplasm

• isomers that do the same function

T or F: in the second step of glycolysis, glucose 6 phosphate is converted into an aldose

F: fructose 6 phosphate is a ketose (rearrangement of carbon 2 and 1 is necessary for cleavage)

Triose phosphate isomerase structure

• TIM barrel

  • helices on the outside and sheets in the middle

  • amino acids held close together

• enzyme represses decomposition of the intermediate

• Glutamate deprotonates Histidine and histidine reprotonates glutamate, followed by histidine deprotonating glutamate and glutamate reprotonating

  • rearrangement to make GAP

The Payoff Phase of Glycolysis

• gaining energy (ATP) instead of using energy

• step 6: (2) GAP to (2) 1,3 bisphosphoglycerate

• step 7: (2) 1,3-bisphosphoglycerate to (2) 3-phosphoglycerate

• step 8: (2) 3-phosphoglycerate to (2) 2-phosphoglycerate

• step 9: (2) 2-phosphoglycerate to (2) phosphoenolpyruvate (PEP)

• step 10: (2) PEP to (2) Pyruvate

step 6 of glycolysis

• 2 GAP to 2 1,3 bisphosphoglycerate

• glyceraldehyde 3 phosphate dehydrogenase (GHAP)

  • adds a phosphate that is in solution to the molecule and 2 molecules of NADH are generated

  • 2 protons are taken from the GAPs in the presence of this enzyme

• oxidation step

step 7 of glycolysis

• (2) 1,3 bisphosphoglycerate to (2) 3 phosphoglycerate

• phosphoglycerate kinase

  • takes one phosphate from 1st carbon and gives to ADP

• 2 ATP generated

step 8 of glycolysis

• (2) 3 phosphoglycerate to (2) 2-phosphoglycerate

• phosphoglycerate mutase

  • phosphate group is moved to second carbon

step 9 of glycolsysis

• (2) 2-phosphoglycerate to (2) PEP

• enolase (uses two waters)

Step 10 of glycolysis

• (2) PEP to (2) pyruvate

• pyruvate kinase

  • takes phosphate group from molecule and gives to ADP

    • 2 ATP molecules generated

• IRREVERSIBLE rxn

more about the oxidation step of glycolysis

• step 6 (glyceraldehyde 3 phosphate (GAP) to 1,3 bisphosphoglycerate)

• oxidation followed by phosphorylation

• oxidation reaction is coupled with dehydration

  • cys is reduced

  • thioester bond is broken and NADH leaves

  • thioester intermediate reduces free energy of the transition state

2 high phosphoryl donors to ATP in glycolysis

• 1,3 bisphosphoglycerate (step 7)

• PEP (step 10)

What is 2,3 bisphosphoglycerate

• intermediate in between step 8

• both carbons briefly phosphorylated

T or F: PEP is higly stable

F: its instability pushes the rxn forward

where does glycolysis occur

in the cytoplasm

what does glycolysis use

• 1 glucose

• 2 ATP

• 2 NAD+

what does glycolysis make

• 2 pyruvate

• 4 ATP (for a net of 2 ATP)

• 2 NADH

  • must be reoxidized to NAD+ for glycolysis to continue

most unfavorable steps in glycolysis

steps 4 (cleavage) and 8 (moving phosphate from 3 to 2)

irreversible steps of glycolysis

• step 1 (glucose phosphorylation)

• step 3 (fuctose phosphorylation)

• step 10 (PEP to pyruvate)

fates of pyruvate

1. anaerobic (fermentation to ethanol)

   1. in fungi

2. anaerobic (2 lactate)

   1. in muscles that are working

3. aerobic (2 acetyl CoA) (citric acid cycle)

fermentation

• regenerates NAD+

• pyruvate is decarboxylated then intermediate (acetalehyde) is made into ethanol

• limiting

• lactic acid can also be fermented by lactate dehydrogenase

  • makes blood more acidic

  • happens in blood to liver

T or F: all sugars follow different pathways to get to pyruvate (they do not all use glycolysis)

F: many types of carbs enter 1 pathway (glycolysis) at different points and use same enzymes

hexokinase vs fructokinase

• hexokinase in fat (adipose tissue)

  • there is a lower quantity in the liver and it has a low affinity

• fructokinase in the liver

• to convert fructose to fructose 6 phosphate (or fructose 1 phosphate if the liver)

galactose as an intermediate

• enters into glycolysis as glucose 1 phosphate

• uridine group has 2 phosphate groups and a glucose

  • high energy transfer of phosphate makes reaction to glucose 1 phosphate favorable

first step of regulation: glucose transport into the cell

• all transporters are 12 transmembrane helices, but they differ in sequence and specificity

• isoforms

• how fast can glucose get into the cell and be converted to glucose 6 phosphate?

GLUT1 and GLUT3

• housekeeping

• in all mammal tissues

• basal glucose uptake

Housekeeping meaning

• maintain basal levels of glucose around a biological system

• high affinity for glucose

• keep glucose around

GLUT2

• in liver and pancreatic B cells

• reacts to signals (insulin)

  • downregulate activity if there’s too much insulin

  • removes excess glucose from the blood in the liver

    • lower affinity for sugar because it’s only active at high concentrations

GLUT4

• in muscle and fat cells

• insulin dependent

• increases if you excercise

  • making sure there’s enough sugar around to make ATP

GLUT5

• in small intestine

• mostly a fructose transporter (not glucose)

Overview of the control of the glycolytic pathway

• hexokinase- traps glucose in the cell

• PFK-1 is the main point of regulation

• Pyruvate kinase regulates payoff phase

PFK-1

• allosteric binding for inhibitors

• tetramer

• active site for fructose 6 phosphate and ATP

PFK-1 in the muscles

• ATP is an inhibitor that bind active site

  • if it binds, causes fructose 6 to buildup and go back to glucose 6 phosphate which inhibits hexokinase

  • inhibited when muscles are at rest

• AMP binds PFK during exercise and stimulates it to make fructose 1,6 bisphosphate

  • this is fed forward to pyruvate kinase, which enhances it

PFK-1 in the liver

• more reliant on hormonal signals like insulin

  • high glucose means high insulin and fructose 2,6 bisphosphate is made

• Fructose 2,6 bisphosphate activates PFK

  • fructose 1,6 bisphosphate is made

T or F: fructose 2,6 bisphosphate is an allosteric signaling molecule that is not part of the glycolysis pathway

true!

T or F: citrate and ATP are inhibitors of glycolysis

True! fructose 2,6 bisphosphate is an enhancer

more on fructose 2,6 bisphosphate

• hormonal enhancer

• like an allosteric protein

• high levels of fructose 2 6 bisphosphate makes amount of fructose 1,6 bisphosphate curve hyperbolic

  • enhancing PFK so more fructose 1,6 bisphosphate is made

  • when ATP is present, curve goes more sigmoidal because of inhibition

regulation of pyruvate kinase in the liver

• post translational modification

• high glucose level leads to dephosphorylation

  • more active

  • fructose 1,6 bisphosphate is an enhancer

• low glucose levels leads to phosphorylation

  • less active

  • ATP and Alanine are inhibitors

    • if there is a high concentration of alanine, pyruvate will be shuttled off to other pathways

muscle control of glycolysis overview

• hexokinase

  • activated by Glucose and insulin

  • inhibited by glucose 6 phosphate

• PFK-1

  • activated by AMP

  • inhibited by ATP (lowers pH) and low pH and citrate and cAMP

• Pyruvate Kinase

  • activated by fructose 1,6 bisphosphate

  • inhibited by ATP and alanine

• the muscle is selfish (either uses glucose or stores it)

• NO fructose 2,6 bisphosphate regulation

Liver and control of glycolysis overview

• glucokinase

  • inhibited by GKRP (keeps in nucleus)

• PFK-1

  • activated by AMP

  • inhibited by ATP, low pH, citrate

• Pyruvate kinase

  • actiavted by fructose 1,6 bisphosphate

  • inhibited by ATP, Alanine, phosphorylation

• focused on the whole body (maintains blood glucose levels)