Biochem Lect 25: Bacteria Cell Wall + Glycolysis Part I

Gram-positive bacterial cell wall

1 bilayer + thick peptidoglycan layer

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**reacts with Gram stain

Gram-negative bacterial cell wall

2 bilayers + thin peptidoglycan layer

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contains lipopolysaccharide

Peptidoglycan structure has 3 major components:

1) tetrapetide (4 amino acids)

2) ɣ-carboxyl linkage

3) glycosaminoglycan disaccharide (NAG-NAM)

What 4 amino acids are in peptidoglycan?

1) L-Alanine

2) Isoglutamate

3) L-Lysine

4) D-Alanine

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(AEKA? LIL D?)

Gram-positive peptidoglycan

contains pentaglycine cross link- makes chain longer!

Gram-negative peptidoglycan

contains direct cross link

Lipopolysaccharide

lipid group + polysaccharide with many repeats attached to outer membrane

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found in Gram negative bacteria

Glycoproteins can be _____ or ______

N-linked, O-linked

O-linked saccharides are attached to OH groups of which amino acids?

serine, threonine, hydroxylysine

N-linked saccharides are attached to amide groups of which amino acids?

asparagine

There are 3 types of N-linked glycoproteins

1) high mannose = mostly mannose

2) complex = many types

3) hybrid = mix of 1 + 2

Louie Pasteur discovered…

pasteurization and glycolysis

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looked at fermentation of grape sugar

Glycolysis

“splitting sugar” to make ATP

Glycolysis is aerobic/anaerobic

anaerobic

products of glycolysis

2 pyruvate, 2 ATP, 2 NADH

2 phases of glycolysis (overall summary)

Phase 1 = glucose → 2 G3P (-2 ATP)

Phase 2 = 2 G3P → 2 pyruvates (+4 ATP, +2 NADH)

Pyruvate has 3 possible fates

1) aerobic = TCA cycle → acetyl-CoA

2) anaerobic = lactic acid fermentation (ex: muscle) → lactate

3) anaerobic in yeast = alcoholic fermentation → ethanol + CO₂

Only _% of glucose goes into glycolysis. 

5

Overall Summary for Phase I 

1) Gluc → G6P = phosphorylation of glucose

2) G6P → F6P = isomerization

3) F6P → F1,6 BP = 2nd phosphorylation

4) F1,6 BP → DHAP + G3P = Cleavage

5) DHAP → G3P = 2G3P

Phase I enzymes

1) Hexokinase/Glucokinase

2) Phosphoglucoisomerase

3) Phosphofructokinase (PFK)

4) Fructose bisphosphate (FBP) aldolase

5) Trios phosphate (3P) isomerase

Phase I Extra

1) -1 ATP (regulated/-ΔG/irreversible)

2) 

3) -1 ATP (regulated/-ΔG/irreversible)

4) 

5) 

Reaction 1

Phosphorylation of glucose

Glucose → Glucose 6 Phosphate

Enzyme = Hexokinase/Glucokinase

-1 ATP

-

priming reaction (-ΔG)!

Step 1 purpose (2)

glucose can easily move in/out of cell, diffuses based on equilibrium

1) phsophorylating glucose traps it inside

2) lots of phosphorylation of glucose → glucose concentration is low so more glucose diffuses into cell

G6P is common to many metabolic pathways such as…

• conversion to glycogen for energy storage (in liver/muscles)

• carbohydrate synthesis

Hexokinase vs Glucokinase shape

Hexokinase = mostly closed → only little change needed for induced fit

Glucokinase = mostly open → lots of change needed for induced fit

Hexokinase vs Glucokinase function/affinity

Hexokinase

• low Km = high affinity for glucose

• found in most tissues

Glucokinase

• high Km = low affinity for glucose

• found in liver 

• only burn ATP when glucose is high !!!!

diabetes → produce low insulin → ___ levels of glucokinase (step 1)

low

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so, cannot handle high glucose in diet and produce little glycogen in liver

(glycogen needs G6P)

Type I vs Type II Diabetes

Type 1 (insulin-dependent) 

• insulin not produced due to early-onset autoimmune dz

• treated with insulin

Type 2 (non-inuslin-dependent)

• insulin produced normally, but insulin-resistant cells

• treated with lifestyle changes

Reaction 2

Isomerization of G6P

Glucose 6 Phosphate → Fructose 6 Phosphate

Enzyme = Phosphoglucoisomerase

Step 2 Purpose (2) 

1) next step is difficult for hemiacetal OH but easy for primary OH

2) activates C3 for cleavage later (?)

Step 3 mechanism

1) open pyranose ring

2) proton removal → ene-diol formation

3) ring closure

*image shows ene-diol

Reaction 3

Phosphorylation of F6P

Fructose 6 Phosphate → Fructose 1,6 Bisphosphate

Enzyme = Phosphofructokinase (PFK)

-1 ATP

-

priming reaction (-ΔG)!

COMMITTED STEP

PFK (Step 3) regulation:

• ATP _____/AMP _____

• citrate _____ (between glycolysis and TCA cycle)

• F2,6BP _____

• ATP inhibits/AMP activates

• citrate inhibits (between glycolysis and TCA cycle)

• F2,6BP activates

PFK binding sites (2) 

• 1 high affinity substrate site

• 1 low affinity allosteric site (inhibitory)

PFK has sigmoidal kinetics at _ ATP and hyperbolic kinetics at _ ATP

high, low

high ATP = low affinity → inhibitory site active →  cooperative behavior (sigmoidal)

low ATP = high affinity → inhibitory site not active →  non-cooperative behavior (hyperbolic)

low ATP increases/decreases AMP due to ____ enzyme

increases AMP, adenylate kinase

F 2,6BP

i) regulator (not intermediate of glycolysis)

ii) created from F6P

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activates PFK - and reverses ATP’s inhibition

(activatate = hyperbolic)

What can reverse ATP’s inhibition?

F2,6BP

increase in glucose → F6P → F2,6BP → ____ PFK affinity. This is feedforward/feedback regulation.

increases, feedforward

Reaction 4

Cleavage

Fructose 1,6 Bisphosphate → DHAP + G3P

Enzyme = Fructose biphosphate aldolase

Step 4 mechanism involves _____ which is a reverse of ______

aldol cleavage, aldol condensation

aldol condensation

ketone attacks aldehyde → aldol

class I vs class II aldolase

class I

• animals

• Lys in active site

• intermediate

• SCHIFF base between carbonyl and lysine = intermediate

class II

• bacteria

• Zn²⁺ stabilized intermediate → no intermediate

Reaction 5

2 G3P

DHAP → G3P

Enzyme = Trios phosphate (3P) isomerase

Step 5 mechanism

• ene-diol formation

• Glu in active site acts as general base

Which enzyme is described as a near-perfect enzyme?

3P isomerase

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works as fast as possible, only limited by diffusion…