Enzymes

Hexokinase

Glucose → glucose 6 phosphate

-1 ATP

Inhibitor: ATP
Co-Substrate: Mg²⁺

Catalysis via proximity effects

Also catalyzes phosphorylation of mannose and fructose

Mg²⁺ Shields - charges of ATP’s alpha and beta phosphate groups making the gamma phosphate more susceptible to Nuc attack by glucose

Phosphoglucose Isomerase

Interconverts G6P (aldose) to ketose (F6P)

Acid Base Catalysis

G6P binds & an acid (Lys) catalyzes ring opening

His imidazole group (base) takes a proton from C2 forming cis-enediolate intermediate

Proton on C1 is replaced & ring closes

Phosphofructokinase

F6P → F1,6BP

Substrates: F6P, ATP

Products: F1,6BP

Activators: ADP, AMP, F2,6BP
Inhibitors: ATP

Catalyzes the rate-determining reaction of glycolysis

-1 ATP

Structure:

Tetrameric Enzyme; each subunit has a substrate and allosteric inhibitor binding site for ATP

• ATP binds equally well to the substrate site regardless of T or R state

• ATP binds to the inhibitor site exclusively in T state

High concentrations of ATP lower F6P binding affinity (shifts graph to right & makes it more sigmoidal)

Activators reverse inhibitory effects of ATP & shift graph back left

T vs R State

R state:

• Stabilized via F6P Binding

• Side Chain of Arg 162 forms an ion pair w/ phosphoryl group of F6P & stabilizes it in the active site

R → T

Protein undergoes a conformational change & a helical turn unwinds/rearranges

• Arg 162 moves away & is replaced by Glu 161

Glu 161 & phosphate group of F6P are both negatively charged, and F6P binds less tightly in the active site due to electrostatic repulsion.

ADP bound at allosteric activator site stabilizes R-state & prevents helical rearrangement associated w/ R→T

Aldolase

F1,6BP → G3P & DHAP

Aldol Cleavage

Non-enzymatic base-catalyzed mechanism

1. F1,6BP binds to the enzyme

2. F1,6BP carbonyl reacts w/ E amino group of active site Lys to form protonated schiff base (iminium cation)

3. C3-C4 bond is cleaved, forming enamine intermediate and releasing G3P

4. Protonation & tautomerization yield iminium cation from schiff base

5. Hydrolysis of iminium cation releases DHAP

Triose Phosphate Isomerase

GAP & DHAP are aldose/ketose isomers

Converted via isomerization reaction w/ enediol intermediate

G3P Dehydrogenase

G3P + Pi + NAD+ → 1,3-BPG + NADH + H+

dehydrogenase, redox reaction

G3P is oxidized

1. G3P binds to the enzyme and is nucleophilically attacked by a sulfhydryl group forming a thiohemiacetal

2. Thiohemiacetal undergoes oxidation to an acyl thioester through hydride transfer to NAD+

3. Pi binds to enzyme-thioester-NADH complex

4. Thioester intermediate is nucleophilically attacked by Pi forming 1,3 BPG

Phosphoglycerate Kinase

1,3 BPG → 3PG

Substrate-level phosphorylation +2 ATP

GAPDH & PGK reactions are coupled

• Consumption of 1,3 BPG (product of GAPDH) in PGK reaction pulls GAPDH forward

• results in delta G of -12.1 kJ/mol

Phosphoglycerate Mutase

3PG → 2PG

Mutase: catalyzes the transfer of functional group from 1 position to another on a molecule

Substrate is positioned close to phosphate group of His 8 on active enzyme

1. 3PG binds to phosphoenzyme in which His 8 is phosphorylated

2. Enzyme phosphate group is transferred to substrate

   1. Intermediate 2,3-bisphosphoglycerate-enzyme complex

3. Enzyme rephosphorlyated by substrate’s 3-phospho group

4. Release of product 2-PG regenerates phosphoenzyme

2-3 BPG inhibits deoxyhemoglobin

Enolase

2PG → PEP

Dehydration

Forms a complex w/ divalent cation (Mg²⁺) before the substrate binds

F^- can inhibit glycolysis by blocking enolase activity

• Forms complex w/ Mg²⁺ at enzyme’s active site

• Leads to 2PG & 3PG build up

Pyruvate Kinase

PEP → Pyruvate

Substrate level phosphorylation +2 ATP

Requires both K⁺ & Mg²⁺

1. Beta-phosphoryl oxygen of ADP nucleophilically attacks PEP phosphorus atom

   1. Displaces enol pyruvate and forms ATP

2. Enol-pyruvate tautomerizes to pyruvate

Tautomerization provides more energy than phosphoryl group transfer

Lactate Dehydrogenase

Pyruvate + NADH + H⁺ → Lactate + NAD⁺

Homolactic LDH catalyzes the oxidation of NADH via pyruvate yielding NAD⁺ & Lactate

1. Pyruvate binds active site of LDH & is stabilized by positive residues (Arg 109 & Arg 171)

   1. Stabilizes & correctly orients pyruvate via electrostatic interactions & H-bonding

   2. NADH must deliver its hydride to a specific carbon in pyruvate (positioning matters)

2. Hydride is transferred from C4 of NADH to C2 of Pyruvate

   1. C4-H bond of NADH is broken

   2. Hydride is transferred to C2 of pyruvate

   3. C=O & e- shifts toward O

   4. NADH becomes NAD+

3. His 195 Base catalysis w/ Pyruvate

4. NAD+ & Lactate are released