Johnson - Metabolism (glycolysis)

Question 1

what is the first step in glycolysis and which enzyme catalyses it?

Answer 1

Glucose + ATP —> glucose-6-phosphate + ADP + H+

catalyzed by hexokinase

Question 2

what is the second step in glycolysis and which enzyme catalyses it?

Answer 2

G6P —> F6P catalyzed by phosphoglucose isomerase

Question 3

what is the third step in glycolysis and which enzyme catalyses it?

Answer 3

F6P + ATP —> F1,6BP + ADP + H+

catalyzed by phosphofructokinase

Question 4

what is the fourth step in glycolysis and which enzyme catalyses it?

Answer 4

F1,6BP —> DHAP + GAP

catalyzed by aldolase

Question 5

what is the fifth step in glycolysis and which enzyme catalyses it?

Answer 5

DHAP —> GAP

triose phosphate isomerase

Question 6

what is the sixth step in glycolysis and which enzyme catalyses it?

Answer 6

G3P + NAD+ + Pi —> 1,3BPG + NADH + H+

catalyzed by glyceraldehyde 3-phosphate dehydrogenase

Question 7

what is the seventh step in glycolysis and which enzyme catalyses it?

Answer 7

1,3BPG + ADP —> 3PG + ATP

catalyzed by phosphoglycerate kinase

Question 8

what is the eighth step in glycolysis and which enzyme catalyses it?

Answer 8

3PG —> 2PG

catalyzed by phosphoglycerate mutase

Question 9

what is the ninth step in glycolysis and which enzyme catalyses it (including its cofactor)?

Answer 9

2PG —> PEP + H2O

enolase and Mg2+

Question 10

what is the tenth step in glycolysis and which enzyme catalyses it?

Answer 10

PEP + ADP + H+ —> pyruvate + ATP

pyruvate kinase

Question 11

where does glycolysis take place?

Answer 11

in the cytosol

Question 12

is O2 required for glycolysis?

Answer 12

no, but oxidation takes place (e- added or lost)

Question 13

is ATP more or less stable than ADP + Pi?

why is this?

Answer 13

ATP less stable

1) -ve phosphate group repel each other
2) entropy is increased (as there are 2 molecules produced from 1)
3) water stabilises ADP + Pi (more interactions between them)
4) free Pi stabilised by resonance structures (isomerism due to e- movement)

Question 14

does the hydrolysis of ATP require energy?

Answer 14

no, it releases energy (under cellular conditions: deltaG = -57kJmol-1)

Question 15

how is ATP used to drive +deltaG reactions?

Answer 15

coupling mechanisms, -ve deltaG reactions drive unfavourable reactions (net free energy must be less than 0 for this to happen)

Question 16

describe the free energy changes involved in the formation of G6P from glucose.

Answer 16

Pi transfer from ATP to glucose has v high Ea in absence of hexokinase.
Uncoupled ATP hydrolysis and Pi transfer to glucose is slow and has a +deltaG
Hexokinase lowers the Ea barrier for direct transfer of the Pi group and avoids hydrolysis

Question 17

how does hexokinase catalyse the formation of G6P?

Answer 17

glucose binding to hexokinase causes conformational change
the hexokinase binding cleft closes (excludes H2O)
this favours direct Pi transfer and not the unwanted side reaction of ATP hydrolysis by water.

Question 18

describe the mechanism of action of hexokinase

Answer 18

hexokinase AS contains Asp which deprotonates C6 ‘OH’ of glucose
deprotonated O:{-} acts as nucleophile and attacks last (gamma) Pi group of ATP
Pi group trasnferred directly to glucose without ATP hydrolysis by water

Question 19

why does the first step of glycolysis happen? (glucose + Pi —> G6P)

Answer 19

G6P contains -ve Pi group meaning it cant cross the cellular membrane. also keeps cellular [glucose] low, which promotes glucose uptake

Question 20

why is triose phosphate isomerase (TIM) used in step 5 of glycolysis?

Answer 20

TIM suppresses formation of toxic intermediate methyl glyoxal from the enediol intermediate (which is the faster reaction in free solution)

Question 21

how does TIM catalyse step 5 of glycolysis?

Answer 21

stops toxic intermediate formation by movement of a 10 a/a loop region over the AS, blocking exit of the enediol until GAP is formed. means it enediol can’t form toxic methyl glyoxal

Question 22

how many molecules of pyruvate are formed per glucose? why is this?

Answer 22

2 molecules of pyruvate, this is because glucose is 6C sugar and forms 2x 3C sugars. This means that steps 6-10 are done twice.

Question 23

describe the 2 step in the oxidation of GAP to form 1,3BPG involving NAD+

Answer 23

oxidation is a 2 step, enzymatically-coupled process:

1) energetically favourable formation of aldehyde to acid
2) energetically unfavourable formation of acyl-phosphate

Question 24

describe the mechanism of action of glyceraldehyde 3-phosphate dehydrogenase

Answer 24

G3PG Cys sidechain reacts with aldehyde in GAP to form hemithioacetal
His side chain removes H{+} from hemithioacetal facilitating its ox. by NAD+ (forms thioester intermediate)
NADH released
Pi group attacks thioester –> 1,3BPG

Question 25

what is substrate level phosphorylation?

Answer 25

the formation of ATP by direct transfer of a Pi group from a high phosphoryl transfer compound to ADP

Question 26

what does having a comparatively high phosphoryl transfer potential mean?

Answer 26

a molecule that has a stronger tendency to donate Pi group than another molecule

Question 27

briefly describe the mechanism of phosphoglycerate kinase

Answer 27

the AS cleft closes around its substrates, excluding water

Question 28

what is the significance of 2PG being isomerised to PEP?

Answer 28

PEP and 2PG have the same metabolic potential energy but PEP is a high-phosphoryl transfer potential compound and therefore releases more potential energy than 2PG when its Pi group is removed.

Question 29

what are the final products of glycolysis?

Answer 29

2 x pyruvate
2 x ATP (4 made, 2 used)
2 x NADH

Question 30

what happens after glycolysis under anaerobic conditions?

Answer 30

further ox of pyruvate can’t occur
this means glycolysis wont proceed for long due to the build up of NADH which cant be recycled in oxidative phosphorylation
therefore NADH used to reduce pyruvate to lactate/ethanol