Unit 3 - Part 2

Persistent hyperinsulinemic hypoglycemia of infancy

caused by mutations that increase glucokinase due to oversecretion of insulin at lower glucose levels

Maturity onset diabetes of the young (GK-MODY)

caused by mutations that slow down glucokinase activity due to reduced retention of glucose at hyperglycemic states

phosphoglucose isomerase

interconverts glucose 6-phosphate < - > fructose 6-phosphate (is a dimer with 2 subunits) and also converts beta glucose into alpha glucose 6-phosphate

During what process does phosphoglucose isomerase turn glucose 6-phosphate into fructose 6-phosphate

glycolysis

During what process does phosphoglucose isomerase turn fructose 6-phosphate into glucose 6-phosphate?

gluconeogenesis

How does phosphoglucose isomerase work?

1. histidine from the other subunit comes into the active site of this subunit to open up the chain by donating H+ to glucose’s ring-closing oxygen

2. E357 withdraws a proton from C2 which causes the e’ to form a pi bonding molecular orbital between C1 and C2

3. the pi bond e’ of C1’s carbonyl bind to a proton to forma. hydroxyl group (becomes dihydroxyenolate intermediate)

4. the proton is returned to H388’ (leaving oxylate form of glucose stabilized by K-519)

5. E357 returns the proton to either C2 to form a new C-H bond (glucose) or C1 to form CH2OH (fructose)

dihydroxyenolate intermediate

temporary molecule that occurs after C2 has lost a proton that allows the carbonyl group to shift from C1 to C2

What does glutamate become when it withdraws a proton?

glutamic acid

What does histidine become when it donates a proton?

imadazolate

What nitrogen is π in H338’?

N1

π in H338’

has H+ attached to it and donates it to glucose’s ring-closing oxygen

Which nitrogen in H338’ is τ?

N3

τ in H338’

is protonated above pH 6

What is the functional group at the #1 carbon of glucose in the linear form?

aldehyde

phosphofructokinase 1 (PFK1)

converts fructose 6-phosphate into fructose 1,6-bisphosphate

Which step is the commitment step to glycolysis?

phosphofructokinase 1

allosteric regulation by ATP/AMP

ATP turns off PFK1 and AMP turns it on

How does ATP turn off PFK1?

at high concentrations, ATP inhibits the connection between the two active sites by binding to the ATP inactivation pocket

How does AMP turn on PFK1?

promotes the connection between the two active sites by organizing the protein structures so that they do form the dimer and then they come together to form the tetramer

What is the structure of PFK1?

4 subunits (a1, a1’, a2, a2’)

What are the substrates for PFK1 and which is a stronger binder?

ATP (stronger binder) and fructose 6-phosphate

How can ATP be both a substrate for PFK1 and also inhibit it?

ATP has a high affinity for the active site but when in excess it spills into the lower affinity allosteric site to inhibit the process

aldol cleavage (using aldolase)

turns fructose 1,6-bisphosphate into dihydroxyacetone phosphate (a ketose phosphate) and glyceraldehyde 3-phosphate (an aldose phosphate)

What is the first step in aldol cleavage?

arginine donates a proton to C5 to open up the ring (serine helps hold fructose 1,6-bisphosphate in place

What happens in aldol cleavage after the fructose 1,6-bisphosphate ring has been opened up?

the nitrogen of lysine makes a shift base bond (imine) with C2 and a H2O molecule is released

schiff base formation

when NH2 (in lysine) reacts with a carbonyl carbon of an aldehyde or ketone to form an imine

imine

functional group with C=N

What happens in aldol cleavage after an imine has been formed by lysine and C2 of fructose 1,6-bisphosphate?

glutamate withdraws a proton from C4’s hydroxyl group, turning the oxygen at C4 into an oxalate

What happens in aldol cleavage after glutamate withdrew a proton from C4’s hydroxyl group?

another lysine stabilizes oxalate and carboxylic acid of glutamate (after it had picked up a proton) and schiff base picks up a proton from solution which draws the electrons from C3-C4 into a C2=C3 double bond

What happens in aldol cleavage after the C3-C4 electrons are pulled into a C2=C3 pi bond?

bond between C3 and C4 is broken, so the electrons from that bond go to C4 to form the aldehyde group

What happens in aldol cleavage after glyceraldehyde 3-phosphate is formed?

glutamate donates back the proton to C3 to form form C-H bond → dihydroxyacetone phosphate

triose phosphate isomerase

converts dihydroxyacetone phosphate into glyceraldehyde 3-phosphate

What portion of glycolysis do you have to start multiplying all products by 2?

trunk portion

What is the difference in eukaryotes and prokaryotes aldol cleavage?

eukaryotes use Schiff base formation to C2 of fructose 1,6-bisphosphate but prokaryotes use a Zn2+ to pull the electrons from a C3-C4 bond to a C2-C3 pi bond

Why do the products have to be multiplied by 2 in the trunk portion of glycolysis?

because triose phosphate isomerase isomerizes dihydroxyacetone phosphate into glyceraldehyde 3-phosphate

glyceraldehyde 3-phosphate dehydrogenase

converts glyceraldehyde 3-phosphate into 1,3 bisphosphoglycerate

Describe the steps of glyceraldehyde 3-phosphate dehydrogenase

1. glyceraldehyde 3-phosphate is oxidized at C1 and the hydride is transferred to NAD+

2. sulfur of cysteine donates a proton to histidine so that cysteine and C1 are able to form thioester bond

3. phosphate (Pi) replaces where the hydride was at C1 on glyceraldehyde 3-phosphate to form acyl-phosphate

4. phosphate from C1 is donated to ADP to make ATP

What amino acids are used in glyceraldehyde 3-phosphate dehydrogenase and what do they do?

cysteine forms a thioester bond with glyceraldehyde 3-phosphate so that it is able to maintain carbonyl and keeps electrons localized in a pi bond and histidine accepts a proton from sulfur of cysteine

thioester bond

between a carbonyl group and a sulfur

What are the investments of ATP in glycolysis?

use of ATP by hexokinase and phosphofructokinase

What are the getting back investments of ATP in glycolysis?

forming of ATP by glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase

phosphoglycerate mutase

a serine picks up a phosphate from C3 of phosphoglycerate and moves it to C2 to convert 3-phosphoglycerate to 2-phosphoglycerate

What is the most conserved enzyme on the planet?

enolase

Enolase

converts 2-phosphoglycerate into phosphoenolpyruvate by the loss of H2O

Describe the steps of enolase in glycolysis

1. arginine donates a proton to 2-phosphoglycerate and takes a proton from lysine

2. lysine takes a proton from 2-phosphoglycerate, which frees up 2 electrons to form a double bond between C1 and C2

3. 2-phosphoglycerate donates a proton to histidine

4. histidine donats a proton to C3-OH which releases H2O

5. this loss of H2O shifts the pi bond to be between C2 and C3

What amino acids are associated with enolase in glycolysis?

arginine, lysine, histidine, and aspartate which holds together two Mg2+ ions

pyruvate kinase

converts phosphoenolpyruvate into pyruvate

Describe the steps of pyruvate kinase

1. phosphenolpyruvate donates a phosphate to ADP to make ATP (leaving pyruvate in enol form)

2. the enol form is rearranged due to the electronegativity of oxygen to the keto form

Is the enol form or keto form low/high energy?

enol form is high energy and keto form is low energy

What is the most exergonic reaction in glycolysis?

pyruvate kinase

What is the ∆G of pyruvate kinase

-7.5 kcal/mol

What are the enzymes in glycolysis to produce pyruvate in order?

1. hexokinase

2. phosphoglucose isomerase

3. phosphofructokinase (PFK1)

4. aldolase

5. triose phosphate isomerase

6. glyceraldehyde 3-phosphate dehydrogenase

7. phosphoglycerate kinase

8. phosphoglycerate mutase

9. enolase

10. pyruvate kinase