Lectures 7-12: Carbohydrate Metabolism & Glycolysis

Glucopyranose formation

Attack of C5-hydroxyl on C1 aldehyde

Six membered hemiacetal ring

Glucofuranose formation

Rotation around C3-C4 → C4-hydroxyl attack

Five membered hemiacetal ring

Acetals

Anomeric OH of the hemiacetal is replaced by an oxygen & ethyl from another sugar molecule → acetal

OH → OEt (or OR) via

Maltose

Disaccharide of 2 glucoses

Linked via α(1→4) glycosidic linkage

OH of glucose converted into OR

Dietary carbohydrates

Starch (2 main components)

Amylose & amylopectin

Amylose

α(1→4) glycosidic linkages

Insoluble - less digestible starch

Forms hydrogen-bonded helices

Yields maltose or maltotriose via amylase

Amylopectin

α(1→4) glycosidic linkages w/ α(1→6) linkages every 25-30 glucose units

Branching increases solubility → more readily digested

Amylase leaves α(1→6) linkages intact → dextrins

(α-)Amylase

Glycosidase or glycoside hydrolase

α-amylase found in animal saliva

Hydrolyses α(1→4) glycosidic linkages randomly along starch chains

Cannot cleave terminal α(1→4) glycosides

γ-Amylase

Glycosidase in small intestine

Hydrolyses remaining α(1→4) & α(1→6) linkages

Glycosidase mechanisms

Acid catalysed partial hydrolysis of acetals into hemiacetals (monosacch)

Leaving group protonated to lower barrier to form oxonium ion intermediate

Neutral nucleophile (H2O) attacks oxonium; reacts like an activated carbonyl

Acidic residue protonates the glycosidic oxygen as it leave the anomeric centre → protonation & C-O breaking happen at the same time

LG blocks lower face of oxonium ion formed → attack gives β-glycoside

Glycosidase (de)protonation limitations

Nucleophile activated deprotonation & LG protonation at the same time is not achievable by modifying the pH of a reaction

Enzymes work via general acid/base catalysis - source/acceptor of protons can activate groups towards reaction not just H+/HO-

Inverted glycosidase mechanism

Changes the stereochemistry at the anomeric carbon

Steric block

Carboxylate side-chain (Asp, Glu) makes nucleophile more reactive - general base catalysis

α-disaccharide (acetal) → β-glucopyranose (hemiacetal)

Retaining glycosidase mechanism

Does not change stereochemistry at the anomeric carbon

Lower face blocked = retention must occur via 2 inversions

HOR lost

Interception of oxonium ion by reaction w/ enzyme

Amylase structure

Accepted long, maybe branched polysaccharide substrates → requires open active site

PDB: 1pig = active site channel for porcine pancreatic α-amylase

Allows oligosaccharide chain to be threaded through rather than a dead end active site pocket (lacks acetal functionality)

Some portion of oligosaccharide binds → unselective cleavage

10^17 fold faster than uncatalysed reactions

Combinations of glycosidases can generate glucose

Glycolysis catalysts

Hexokinase

Phosphoglucose isomerase

Phosphofructokinase

Aldolase

Triose phosphate isomerase

Glyceraldehyde-3-phosphate (G3P) dehydrogenase

Phosphoglycerate kinase

Phosphoglycerate mutase

Enolase

Pyruvate kinase

Glycolysis overview

10 enzyme catalysed reactions to convert 1 glucose molecule into 2 pyruvate molecules

1 glucose (6C) → 2 pyruvate (3C)

Steps up to G3P dehydrogenase are ATP consuming

Glycolysis is overall ATP generating

Steps after G3P occur twice per glucose molecule consumed

4/10 steps involve phosphorylation via kinases

ATP = phosphate source

Adenosine triphosphate (ATP)

Can transfer its terminal phosphate group

Forms adenosine diphosphate (ADP)

Phosphoanhydride linkages (P-O-P)

High energy bonds b/c they are weak (easy to break) & formation of stronger bonds in the products is exergonic

ATP → ADP

1Electrostatic repulsion of negative charges destabilises phosphoanhydrides

2 Resonance stabilisation of negative is less in phosphoanhydrides

3 Hydrolysis of P-O-P results in more particles

4 Solvation effects

Enthalpy changes in 1, 2, & 4

Entropy changes in 3 & 4

Step 1: hexokinase

Kinase-catalysed phosphorylation of alcohols

Nucleophilic attack at phosphorus

Aspartate side chain of hexokinase acts as base to increase nucleophilicity

Mg2+ = cofactor needed to complex ATP, increasing electrophilicity & reduces negative charge on phosphates (charge repulsive to nucleophile)

Active site of enzymes using ATP often have side chains w/ positive charge

ATP → ADP

Step 7: phosphoglycerate kinase

Kinase-catalysed dephosphorylation

Carboxylate is better LG than alkoxide

Transfer of phosphate group from 1,3-biphosphoglycerate to ADP

Mg2+ coordinates substrate & ADP; counteracts electrostatic repulsion

Asp coordinates Mg2+

Positively charged lysine side chains

ADP → ATP (production)

Kinase form & function

Mg2+ ion (cofactor) complexation w/ negatively charged phosphate groups

Many weak interactions combine for big effect ie. hydrogen bonds

H-bonding to ribose -OH in ATP, substrate -OHs, phosphate oxyanions

Step 10: pyruvate kinase

Phosphoenolpyruvate dephosphorylation

1st Mg2+ ion coordinates ADP

2nd Mg2+ ion coordinates a water molecule making it more acidic & stabilising HO-

Full enzyme activity requires a molecule of FBP (allosteric regulation effect)

ATP → ADP

Step 3: phosphofructokinase

Phosphorylation of α-fructose-6-phosphate (F6P) to β-fructose-1,6-biphosphate (FBP) & change in stereochemistry at anomeric position

F6P anomers equilib via open chain form of ketohexose

α→β stereochemistry switch (inversion)

Phosphorylation

Isomerisation

Transformation of a molecule into a different isomer

Cis & trans conformations

Catalysed by isomerases - starting materials & products have same formula

Enols & enolates

Aldehydes & ketones are in equilib w/ small amounts of enols → alkENe alcohOLs; 1 in 10^7 in enol form for simple isolated systems

Enzymes accelerate enol formation (cannot change K) by facilitating proton transfer

Equilib constant K = 10^-7 for acetaldehyde

1,3-dicarbonyls enol formation K = 0.3; effective delocalisation provides more payoff for sacrificing the stronger C=O (change in double bonds)

Enols are nucleophilic at α-carbon

Enolates are negatively charged & more nucleophilic

Interconverting keto/aldo & enolate forms

Carbonyls act:

Electrophilic at C in C=O

Nucleophilic at α-C in C=C-OH

α-Hydroxycarbonyls

Enediols = carbonyls w/ hydroxyl group at α-carbon

Different R1 & R2 → α-hydroxycarbonyls interconverted via enediolate

Step 5: triose phosphate isomerase

Dihydroxyacetone phosphate (DHAP) → G3P (ΔG>0)

Only H on lower face removed

Step 2: phosphoglucose isomerase

G6P (hemiacetal) → enediol(ate)

Open chain form via co-op reaction of acidic (His) & basic (Lys) residues

Isomerisation of aldohexose glucose (C1 carbonyl) → ketohexose fructose (C2 carbonyl)

C2 deprotonated by glutamate residue → enediolate (in glucose & fructose)

Other end of enediolate reprotonated → aldose/ketose interconversion

Isomerase moves hydrogen from C1 to C2 → open chain form of F6P

Elimination reaction

Dehydration reactions (removing H2O)

2-phosphoglycerate → phosphoenolpyruvate

E1 = elimination, unimolecular - 1 molecule involved in rate determining step

Tertiary carbocation = relatively fast

E2 = elimination, bimolecular - 2 molecules involved in rate determining step

E1cb mechanism: unimolecular elimination via conjugate base

Step 9: enolase

Elimination of H2O (- H2O) from 2-phosphoglycerate via E1cb mechanism

Enolate = conjugate base, BH = conjugate acid

Fast, reversible deprotonation of acidic C-H-α to the carboxyl group

Mg2+ helps accommodate multiple negative charges

HO- leaves

Unreactive → reactive

Aldol reactions

Reaction of an enol or enolate w/ a carbonyl → β-hhydroxycarbonyls

Carbonyl = electrophilic & enol = nucleophilic

Retro-aldol reaction

Reverse process of aldol reactions

β-hydroxycarbonyl collapses to a aldehyde/ketone & enol/enolate

Catalysing requires acid (carbonyl→enol) & base (alcohol→carbonyl)

Step 4: aldolase

Retro-aldol reaction catalysed by aldolase on open chain FBP

Open chain FBP (-H+) → hemiaminal → G3P or enamine → DHAP

Positively charged iminium accepts electrons of broken C-C bond

Lysine reacts w/ ketone

More reactive than carbonyl (allows retro-aldol)

FBP → G3P and/or DHAP

Class II aldolases

Algae, fungi & some bacteria

Breaks C-O → opens sugar

Uses Zn2+ as Lewis acid to stabilise formation of enolate by forming a zinc enolate

Alcohol & ketone attached to carbon breaks bond

Class I aldolases

Plants & animals

Use a lysine -NH2 to form an iminium ion (covalent catalysis)

More reactive in retro-aldol reaction

Step 6: G3P hydrogenase

Oxidises G3P → 1,3-bisphosphoglycerate → + NAD+ & Pi, - NADH/H+

Base obstructs hydrogen

G3P → deprotonated hemithioacetal (hemiacetal but O→S) via cysteine-SH

H- results in a thioester intermediate bound to enzyme

Thioester cleaved by nucleophilic attack at carbonyl by phosphate (PO4 3-) → acyl phosphate

NAD+ ←→ NADH

H- formally lost is accepted by NAD+ → NADH (easily interconverted)

Hydride/H- = highly reactive & poor leaving group; requires hydride acceptor

Mutases

Subset of isomerases

Transfers functional groups (phosphate)

Step 8: phosphoglycerate mutase

3-phosphoglycerate → 2-phosphoglycerate

Phosphorylates position 2 then dephosphorylates position 3

Phosphorylation via phosphorylated histidine-8 & glutamate-86 as base

Rotates & same histidine dephosphorylates position 3

Histidine phosphorylates next 3-phosphoglycerate w/ same phosphate

Reversible glycolysis steps

k = 1 in most reversible steps → concentrations influence course of reaction

Steps, 2, 4, 5, 6, 7, 8 & 9 (all except 1,3 & 10)

Start & end of process are tightly controlled

Irreversible glycolysis steps

Step 1: hexokinase - phosphorylation of glucose → G6P

Step 3: phosphofructokinase - phosphorylation of F6P → FBP

Step 10: pyruvate kinase - phosphoenolpyruvate dephosphorylation

ATP consumed in 1&3 makes them highly exergonic (large negative ΔG)

Formation of pyruvate in 10 is highly favourable & allows ADP→ATP to form

Glucose to G6P (step 1)

Quick & irreversible phosphorylation of glucose

Minimises intracellular glucose → ensures concentration gradient for inwards transport is maintained

Prevents transport out of cell → G6P is not a substrate for glucose transfer proteins - phosphate group disrupts interactions

Prevents diffusion from cell - charged G6P cannot diffuse through phospholipid membrane

Phosphorylation is coupled to favourable reaction ATP→ADP (exergonic) - release of electrostatic repulsion in triphosphate

Hexokinase is inhibited by G6P → high G6P = glucose uptake reduced

F6P to FBP

Also coupled to ATP→ADP

Phosphorylation at C1 & C6 hydroxyls of C6 sugar allows both products from aldolase catalysis to be phosphorylated → cannot leave cell

FBP = allosteric activator for downstream enzyme pyruvate kinase

Feed forward or opening the floodgates

Formation of FBP builds G3P concentration (& via DHAP) & feeds into sequence of equlibria

Allosteric modulation of pyruvate kinase = means to escape that process & prevent build up

Phosphoenolpyruvate (PEP) to pyruvate

Pyruvate = important intermediate in many metabolic processes

Concentration & rate of formation is carefully regulated

FBP feed forward ensures steady metabolic flux through equilibria

Gluconeogenesis

Biosynthesis of glucose, reverse process of glycolysis

Irreversible steps are different to overcome unfavourable ΔG

Different reactions = separate regulatory pathway (prevents futile cycle)

Start & end of process are also tightly controlled

Shares same reversible steps, different reactions ensure favourable ΔG

Gluconeogenesis step 1a: pyruvate carboxylation

Pyruvate (3C) → zinc enolate → oxaloacetate (4C)

Pyruvate enolate reacts w/ CO2 in aldol type reaction

Deprotonates alpha to carbonyl to form zinc enolate

CO2 = electrophilic carbonyl component of aldol reaction

CO2 (1C) attaches to zinc enolate (3C) → oxaloacetate (4C)

CO2 generation

HCO3- → CO2

Activated w/ ATP → carboxyphosphate formation

Trapped by biotin (cofactor) → N-carboxybiotin

N-carboxybiotin liberates CO2

Oxyanion stabilised by Lys-NH3+

LG ability improved - Pi + H+ is better than Pi

Gluconeogenesis step 1b: PEP carboxykinase

Catalyses standard β-ketoacid decarboxylation of oxaloacetate w/ phosphorylation of pyruvate enolate by guanosine triphosphate (GTP)

CO2 removed from oxaloacetate via entropy & acid or other electrophiles

Electrons from carboxylate/acid form relatively stabilised enolate

GTP phosphorylates oxaloacetate

Mg2+ assists w/ organisation & multiple negative charges

Adding + losing CO2 uses ATP & GTP = favourable energy release

Gluconeogenesis step 2: fructose-1,6-biphosphatase

Hydrolyses phosphate at C1 of FBP

ATP formation from FBP = too energetically costly → Pi lost instead

2 phosphates → 1 phosphate via Glu, Asp & Mg2+ or Mn2+

Gluconeogenesis step 3: glucose-6-phosphatase

Dephosphorylation of G6P → glucose

Phosphate is transferred to a histidine

Hydrolysed in water

Active site reactivated