Biochemistry Unit 3

Metabolism

Overall process through which living organisms aquire and use free energy to carry out various properties

Catabolism

Degredation; nutrients and cell constituents are broken down to salvage their components and/or make energy avaliable

Anabolism

Biosynthesis; biomolecules are synthesized from simpler components

Metabolic pathways

Series of connected enzymatic reactions that produce specific products

Metabolites

The reactants, intermediates, and products in a metabolic pathway

How is free energy obtained in metabolic pathways

Complex metabolites exergonically broken down into simpler products

Free energt released in the degradative process is conserved through teh creation of different molecules ATP from ADP and Pi, or

Free energy storage

Stored through synthesis of ATP from ADP + Pi or by the reduction of a coenzyme such as NADP+ to NADPH.

Major energy sources for anabolic reactions

Generalizations about metabolism

Metabolic pathways are interconnected and converge into common hubs

All pathways occur simultaneously

Facilitated by enzymes

Regulated

Main source of fuel for many metabolic pathways

Glucose

Transportation of metabolites in tissues

Only glucose is transported through blood

There are other specialized ways/functions to transport

Where specifically does metabolism occur?

Primarily in cellular compartments, primarily the cytosol and mitochondrion

Lysosome has other related processes that occur, but not necessarily metabolic pathways

Why are enzymes important in metabolism

They are biological catalysts, that allow greater reaction specificity, higher reaction rates, milder reaction conditions(don’t need/cause intense conditions that may kill cell to occur), and capacity for regulation

What do enzymes sometimes need?

Cofactors

What is a cofactor

a non-protein chemical compound or metallic ion required for an enzyme to function as a catalyst in a biochemical reaction

Cofactors are the vitamins and minerals from your diet.

How are enzymes highly specific

through binding of active site of enzyme by lock and key model

Have geometric complementarity and electronic complementarity for substrate/active site

This means substrate physically fits into active site, while having favorable electronic charges/residues that bring them together

What are the electronic components of enzyme

The residues of the active site;

1) hydrophobic effect- allows substrate to bind to active site and displace water, increasing entropy, which is favorable(don’t compete with H2O for reaction)

2)Hydrogen bonding

3)Ion-dipole

How do enzymes change reaction kinetics

Enzymes DO NOT change thermodynamics(delta G remains the same), but rather lowers the activation energy (X double cross/ delta delta G) to make reaction more attainable energetically

Example of how enzymes change the rate of reaction

in proteases, they contain an oxyanion whole which lowers the transition state energy

What type of environment to the top and bottom histidines have in common?

Hydrophobic environment(promotes initial status of unprotonated/uncharged)

Hydrophilic envirnment (promotes charges, polar initial status)

Covalent catalysis

Substrate is temporarily covalently bonded to active site of enzyme

Nucleophilic groups can catalyze reactions through transient formation of covalent bonds with the substrate

Oxonium ion covalent catalysis

Cofactor subcatagories

Metal ion: may play a role in stabilizing enzyme structure, or directly in catalysis

Coenzymes: organic molecules

• Cosubstrate: associated transiently

• Prosthetic group: permanently associated with enzyme, often through covalent bond

Oxidoreductase

Enzyme for oxidation-reduction reaction

Transferase

enzyme that aids in transfering of functional group

Hydrolase

Enzyme that helps in hydrolysis reactions

Isomerase

Enzyme that helps in isomerization reactions

Ligase

Enzyme that helps in bond formation reactions

Kinase

Enzyme that uses ATP to add a phosphate group to molecule

Dehydrogenase

Any enzyme in a oxidation/reduction reaction that consumes NAD-/NADH

Overview of thermodynamics of reaction

deltaG>0

Non-spontaneous/endergonic

deltaG=0

Reversable and at equilibrium

deltaG<0

Spontaneous/exergonic

How can direction of chemcial reactions be determined?

Le Chatelier’s principle; tells us how equilibrium shifts

Most metabolic pathways are in equilibrium

What is the consequence of reactiions functioning far from equilibrium

These irreversible reactions confer directionality on that pathway(push pathway in equilibrium in one direction)

Commit metabolites to pathway(thin Le chatleier’s)

These are the rate limiting reaction and serve as potential control points for the pathway(allows enzyme regulation)

Reaction far from equilibrium impacting metabolic pathway example

Exergonic/spontaneous reaction essentially converts all reactants into products

These products are reactants for the second step of pathway; high concentration of step 1 product/step 2 reactant pushes step 2 towards products, confering directionality of the pathway

How do we calculate free energy of pathway?

We can calculate the changes in free energy of an entire pathway by adding the changes in free energy of each reaction within that pathway.

How is enzyme activity controlled (short term;seconds to minutes)

Allosteric Activation/inhibition

Covalent modification-Phosphorylation and hormones

Substrate cycle-two metabolic pathways run simultaneously in opposite directions

How is enzyme activity controlled (long term; hours to days)

Genetic control- regulation of enzyme availability

Allosteric control

Binding of moleucle to non-active site that conformationally changes enzyme

Converts enzyme between T state and R state

Binding of desired substrate makes enzyme more active

How can metabolic pathway products serve as effectors and why can this be beneficial?

High concentration of product indicates there is a plentiful amount, and more production would waste energy; if product serves as an inhiitor upstream of pathway, it can self regulate itself, and stop the metabolic pathway from reducing more/at the rate it is doing so

Vice versa for activators, except high concentration of reactants serve as activators downstream

Gluconeogenesis(what is it and delta G)

Synthesis of glucose

reverse reaction

deltaG«0

glycolysis(what is it and delta G)

Degradation of glucose

Forward reaction

deltaG«0

How can gluconeogenesis and Glycolysis both have a -delta g if they are opposite reactions?

Do this by having two different sets of enzymes to control either direction, and take control by activating/inactivating any given enzyme

**equilibrium steps only use one enzymes, leaving them in equilibrium

Why is metabolism stepwise?

so energy can be stored for other processes , and not be used all at once/lost to heat

How is energy generated in metabolism stored?

In energy packets that readily release/generate energy for various processes

ATP, Ac-CoA, NADH, FADH2

What are different bonds in ATP?

Most common/accesible reaction with ATP

Breaks one phosphoanhydride bond

Phosphoanhydride vs phosphoester free energy

Phosphoanhydride bond is less stable, leading to its hydrolysis releasing more free energy

ATP hydrolysis reactions and standard free energy

Why is phosphoryl-transfer so favorable?

1. Resonance stabilization is greater in the products.

2. Electrostatic repulsion is reduced after hydrolysis.

3. Hydrolysis products are better solvated.

Why is phosphoryl transfer so useful?

The free energy change associated with phosphoryl group transfer from ATP is negative enough to be coupled with other metabolic reactions and power these reactions

How do we make this endergonic reaction proceed?

Couple reaction with ATP/ATP hydrolysis

What is the delta g of this reaction (deltaG reaction 1=+13.8, deltaG reaction 2= -30.5)

thioesters use in energy storage

Thioesters can conserved/act like ATP, in that it saves energy rather than it being lost as heat

Has a similar -deltaG to ATP

NADH

Electron acceptor; can accept two electrons in the form of H —

NADH functions

NAD+ (derived from niacin) is a cosubstrate for many dehydrogenases. NAD+ accepts electrons and transfer them to other compounds.

NADH can shuttle electrons to the Electron Transport Chain (ETC).

FADH2

Can accept two electrons but one at a time (in the form of radical H)

FADH2 functions

FAD(derived from vitamin B2 ) is a cosubstrate for some dehydrogenases.

FAD accepts electrons and transfer them to other compounds.

FADH2 can shuttle electrons to the ETC

Substrate-level phosphorylation

The transfer of a phosphoryl group from a phosphorylated substrate to ADP to regenerate ATP.

Oxidative phosphorylation

Process by which energy released by electrons is used to generate ATP.

Digestion of carbohydrates

1)Salivary amylase begins digestion in mouth

2) salivary amylase is denatured by stomach acids

3)Pancreatic emylase continues digestion in small intestine

4) Enzymes in microvilli break down carbohydrates to monosaccharides

5)Resistant starches and fibers are digested and fermented in the large intestine and colon

What is the basic mechanism of polysaccharide breakdown in low pH envirnment

Where do energy generating processes occur?

Within the cell; specifically, the cytosol and mitochondria

How is glucose brought into the cell and what is done to it innitialy?

Via GLUT’s (Glucose transporters) which bring it in from across the plasma membrane

Once inside, it is phosphorylated

Phosphorylation locks glucose inside the cell

Why does phosphorylation lock glucose inside the cell

The large and polar phosphate group prevents the glucose from crossing the membrane barrier

Rate limiting steps

Steps that are far from equilibirum and have a very negative delta g

General overview of glycolysis

hasd two stages stage 1 and stage 2

10 enzymatic reactions, and converts glucose into two pyruvates

net gain of 2 ATP and 2 NADH

Stage 1 general overview

5 steps

requires in investment of 2 ATP’s

Stage 2 general overview

Energy recovery phase

5 steps

Produces 4 ATP and 2 NADH

Step 1 of glycolysis

Hexokinase phosphorylates glucose at C6

Coupling with ATP hydrolysis makes reaction favorable

Substrate: Glucose

Cofactors: Mg2+, ATP

Enzyme: hexokinase

Very negative delta G! (-27kJ/mol—> Favorable)

Half reactions of step 1

Glu+ Pi—> Glu-P

ATP—> ADP + Pi

Adds up to

Glu+ATP —> Glu-P +ADP

Hexokinase enzyme

Preforms step 1 of glycolysis

Relatively nonspecific and will phosphorylate many different sugar molecules

This step is what locks glucose inside of cell

What is function of Mg2+

Cofactor; stabilizes negative charge of ATP, shielding these negative charges

Also makes the y-phosphorous atom(third one) more accessible to nucleophilic attack by C6-OH of glucose

Why is C6-OH attacked?

It is the only primary alcohol in glucose, making it the most reactive

Step 1 mechanism

Water is a nucleophile that attacks one of the phosphate groups in ATP

Example of hydrophobic effect at active site with H2O being replaced to prevent h2o from interrupting the reaction

enzyme works by excluding water and prevents water from acting as a nucleophile and competing with glucose/ATP reaction

Glucose binding to hexokinase

Substrate binding induces conformational change of enzyme.

Enzyme closes on substrate (like a baseball glove).

Excludes H2O from active site.

Brings substrate and cofactors into close proximity.

Step 2 of glycolysis

G6P converted into F6P; catalyzed by phosphoglucose isomerase

Enzyme: phosphoglucose isomerase (PGI)

Phosphoglucose isomerase mechanism

Works by using general acid–base catalysis. It first opens the glucose ring, then shifts the carbonyl from C1 to C2 through a cis-enediolate intermediate, with active-site acids and bases moving protons back and forth. Finally, it closes the ring into the five-membered fructose form and releases F6P.

Step 3 of glycolysis

F6P phosphorylated into FBP

Cofactors: ATP, Mg2+

Enzyme: phosphofructokinase(PFK); similar traits to that of hexokinase

Very negative delta g(ATP hydrolysis coupled reaction)

What are the two forms of F16BP that exist and which is the product of a reaction and which is the reactant in a reaction

These forms are in equilibrium with eachother and regularly interconvert.

Step 3 product is FBP cyclic form and step 4 reactant is linear/non-cyclic form

Why does step 4 use FBP isomer?

Aldolase needs a free C2 ketone, which only the open-chain form has. An active-site lysine attacks that carbonyl to form a Schiff base, and that iminium pulls electrons so the C3–C4 bond can break, splitting FBP into DHAP and G3P. In the ring form, C2 is tied up in the ring and isn’t a carbonyl, so this mechanism can’t happen.

Step 4 of glycolysis

Aldolase cleaves FBP

FBP—> DHAP + GAP

6 C sugar converted into two 3 C molecules

Substrate: Open chain form of FBP

Enzyme: Aldolase

Product: GAP + DHAP

Step 4 mechanism details

Aldolase catalyzes the aldol cleavage between C3 and C4.

Requires a carbonyl at C2 and hydroxyl at C4.

Using step 4 reaction, why is isomerization of G6P necessary?

If glucose is not isomerized, it would form a 4 C and 2 C molecule, which is not ideal for creating the same 3 C molecule, which is eventually done in glycolysis

Isomerization allows for aldolase to effectively split the substrate in half into two 3 C molecules

Aldolase mechanism

Lysine attacks carbonyl forming a schiff base making it more electrophilic, and Asp attacks C4-OH as a basewhich effectively leads to C3-C4 bond cleavage

This schiff base must be hydrolyzed to remove enzyme(attached via covalent catalysis)

Why is lysine the only residue that can be used in aldolase

Aldolase’s mechanism requires a Schiff base, which can only form from a primary amine attacking the carbonyl, and lysine is the only amino acid with a primary amine side chain. Other nitrogen-containing residues like arginine, histidine, asparagine, and glutamine aren’t nucleophilic enough to do this. The active site also lowers lysine’s pKa so it stays neutral and able to attack.

Step 5 of glyvolysis

Triose phosphate isomerase converts DHAP into GAP

DHAP—> GAP

substrate: DHAP

enzyme: triose phosphate isomerase

Product: GAP

triose phosphate isomerase mechanism notes

Acid-base catalyzed isomerization.

Maintained at equilibrium.

Triose phosphate isomerase helps make enediol intermediate

GAP continues down path, DHAP converted to GAP as GAP is consumed.

First stage of glycolysis overview

1 Glucose converted to 2 GAP and 2 ATP used

What are high energy compounds

Compounds whose delta G has a large magnitude, negative delta G

They are used to help form ATP via ADP and Pi

Step 6 of glycolysis

GAP, NAD+, and Pi converted into 1,BPG (high energy intermediate), NADH, and H+

Catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Redox reaction with dehydrogenase enzyme; aldehyde is oxdized and NAD+ is reduced

Enzyme: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Substrate: Glyceraldehyde-3-phosphate (G3P)

Cosubstrates: NAD⁺ (oxidizing agent) and inorganic phosphate (Pi). No ATP is used.

GADPH mechanism

Pi is a cosubstrate

Keep note that Cys is catalytic residue and needs a base to deprotonate it in order to attack aldehyde; Represents a transient covalent bond, and will serve as a good LG(TS energy).

Role NAD+ has as a oxidizing agent and pulling off aldehyde H, and allowing for Pi to attack

Step 7 of glycolysis

1,3BPG+ADP converted into 3PG + ATP; catalyzed via PGK

Enzyme: Phosphoglycerate kinase (PGK). It’s named for the reverse reaction, where ATP phosphorylates 3-phosphoglycerate.

Substrate: 1,3-bisphosphoglycerate (1,3-BPG)

Cosubstrate: ADP, bound as a Mg²⁺–ADP complex (Mg²⁺ is the required metal cofactor)

Products: 3-phosphoglycerate (3PG) and ATP

Why does step 7 reaciton work

Look at the delta G’s of the reaction; releasing a Pi group from 1,3BPG is highly favorable, and can be coupled with formation of ATP which isn’t favorable

1,3-BPG + H₂O → 3PG + Pi ΔG°′ = −49.3 kJ/mol

ADP + Pi → ATP + H₂O ΔG°′ = +30.5 kJ/mol

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1,3-BPG + ADP → 3PG + ATP ΔG°′ = −18.8 kJ/mol

Step 7 mechanism

Step 8 of glycolysis

3PG converted to 2PG via PGM (phosphoglycerate mutase)

Substrate: 3PG

Product: 2PG

Enzyme: phosphoglycerate mutase (PGM)

Mutase

An enzyme that catalyzes the transfer of a functional group from one position to another on a molecule.

Step 8 Simple Mechanism

PGM mechanism

His acts as intermediator for Pi transfer

Acidic amino acid is also needed for transfer of original Pi onto the His