Chapter 1 and 2/13: Part 2

What is PEP?

Phosphoenolpyruvate, a molecule with a very high phosphoryl transfer potential

Where does PEP fit into metabolism?

PEP is an intermediate in glycolysis, near the end of the pathway

Why is PEP important in glycolysis?

PEP has enough phosphoryl transfer potential to transfer its phosphate to ADP and make ATP

What type of ATP production occurs when PEP transfers its phosphate to ADP?

Substrate-level phosphorylation

PEP + ADP → pyruvate + ATP

How many of glycolysis’ 4 gross ATP are produced using PEP? Where do the other 2 gross ATP in glycolysis come from?

2 ATP

From 1,3-bisphosphoglycerate (1,3-BPG) transferring a phosphate to ADP

What are the products of glycolysis?

2 ATP from 1,3-BPG

2 ATP from PEP

= 4 gross ATP

Does the PEP reaction simply remove a phosphate from PEP?

No. The molecule also undergoes a major structural change: PEP’s enol form converts into pyruvate’s keto form

Why is the standard free energy of hydrolysis for Phosphoenolpyruvate (PEP) significantly larger than that of ATP? in other words, why is the PEP → pyruvate reaction so favorable?

Because the reaction produces pyruvate in its keto form, which is much more stable than the enol form in PEP

What does “more stable” mean in terms of energy?

A more stable molecules is in a lower-energy state, meaning it has less potential energy

Why does PEP have such a high phosphoryl transfer potential?

PEP is “trapped” in a less stable, higher energy enol form. After phosphate transfer, it can convert into the much more stable keto form of pyruvate

What happens to PEP after it loses its phosphate?

It initially forms an enol form of pyruvate, which then rearranges into the more stable keto form

• PEP → enol pyruvate → keto pyruvate

Why can PEP make ATP so effectively?

PEP starts in a high-energy, unstable state. When it transfers its phosphate to ADP, it becomes pyruvate, which can rearrange into a much more stable, lower-energy keto form. This large energy difference helps drive ATP formation

Why does the enol → keto conversion help drive ATP formation?

The conversion releases the stored potential energy associated with the unstable enol form, making the overall reaction strongly favorable

What structural feature of 1,3-bisphosphoglycerate (1,3-BPG) contributes to its high phosphoryl group transfer potential?

The hydrolysis product, 3-phosphoglycerate, is stabilized by resonance around the carboxyl carbon which locks carboxyl into one form

How does the ionization of $3$-phosphoglyceric acid into $3$-phosphoglycerate affect the hydrolysis of $1,3$-BPG?

It removes a reaction product, favoring the forward hydrolysis reaction.

What does “removal of 3-phosphoglyceric acid by further metabolism” mean?

It means that 3-phosphoglyceric acid (3-PG) gets used in the next reactions of glycolysis, so it doesn’t build up

Why does removing a product of 3-phosphoglyceric acid favor the forward reaction?

If the product is continually removed/used up, the reaction has less incentive to go backward, so it is pushed toward making more product. Going backward may require additional energy or an additional reaction step, making the reverse direction less favorable

What is the primary factor that stabilizes the product of phosphocreatine hydrolysis, creatine?

Resonance stabilization of the creatine molecule and the release of Pi

Why are most oxygen esters more stable than acyl phosphates or thioesters?

They can form resonance structures around the carbonyl carbon and the oxygen atom

Why are thioesters, such as Acetyl-CoA, more reactive than regular oxygen esters?

Sulfur does not readily form double bonds with carbon, leading to much less resonance stabilization

Why is hydrolysis of an acyl bond favorable?

The products formed after hydrolysis are more stable and have lower free energy than the starting acyl compound

What is thioesters?

A thioester is a compound in which a sulfur atom replaces the oxygen atom in an ester linkage, resulting in a C–S–C=O structure instead of the typical C–O–C=O found in oxygen esters.

Why is there a greater difference in free energy between reactant and products for thioesters?

Thioesters undergo much less resonance stabilization than oxygen esters

Phosphorylation potential, ΔG_p is?

The actual free energy of hydrolysis of ATP under intracellular conditions which varies from cell to cell and over time based on [ATP, ADP, P_i, etc]

How does ΔG_p usually compare to the standard free-energy change ΔG° for ATP hydrolysis in vivo?

ΔG_{p is usually greater than the standard free-energy change,} ΔG° because cellular conditions ≠ standard conditions

What is the importance of cleaving the gamma-beta bond cleavage in ATP?

The transfer of a phosphate group to power cellular anabolism

Bond energy release

Is phosphate the only group that can be transferred from ATP?

No. AMP or PPi (pyrophosphate) can also be transferred

What is pyrophosphate (PPi)?

Two phosphate groups linked together

What determines which group is transferred from ATP? What is generally transferred from ATP in these reactions?

Which phosphate bond is attacked during the reaction

AMP

Adenylylation

A reaction that transfers an adenylate (5'-AMP) group from ATP to a substrate, often used for energy coupling that is thermodynamically very favorable

What happens to PPi when it is produced as a byproduct?

It is hydrolyzed into two inorganic phosphates (2 Pi)

What byproduct of adenylylation is hydrolyzed by inorganic pyrophosphatase to drive reactions forward?

Pyrophosphate (PPi)

Hydrolysis of the _______ bond releases somewhat more energy than hydrolysis of the ___ bond

a-B phosphoanhydride

B-y bond

How many high-energy phosphate bond cleavages are typically required to form an acyl-P intermediate during adenylylation?

Two cleavages (one to release PPi and one to hydrolyze PPi).

The energy for peptide bond formation in protein synthesis initially comes from the _____.

Charging of tRNA with an amino acid.

During the charging of tRNA, what is the intermediate form of the amino acid before it is transferred to the tRNA?

Aminoacyl-AMP (a.a.-AMP)

In cytosolic ATP synthesis, the transfer of a phosphoryl group from a donor with higher transfer potential to ADP is called _____.

Substrate-level phosphorylation

Which two compounds in the glycolysis pathway serve as donors for substrate-level phosphorylation?

1,3-bisphosphoglycerate (1,3-BPGA) and Phosphoenolpyruvate (PEP).

What type of intermediate is often formed when an acyl group is transferred?

A high-energy acyl intermediate

What are the two possible high-energy acyl intermediates?

R-AMP (acyl-AMP) or R-PO₄ (acyl phosphate)

What determines the type of bond formed with the acyl group?

The nucleophile that attacks the acyl group

What bond forms if the nucleophile is: N, O, S?

N: amide bond

O: ester bond

S: thioester bond

What happens to the acyl group after forming a high-energy intermediate?

It can be transferred to another molecule, forming a new bond

Through group transfer reactions, ATP provides the energy for:

• The synthesis of informational macromolecules (DNA, RNA, protein)

• The transport of molecules and ions across membranes against gradients

Through hydrolysis, ATP provides the enrgy for:

Muscle contraction

Why is acyl group attacked by NH2?

To form peptide bond and become more stable and able to resonate

Most phosphoryl donors for ATP synthesis are generated via _____ reactions.

Redox (oxidation-reduction)

What enzyme is responsible for transferring phosphoryl groups between $ATP$ and other nucleoside diphosphates ($NDPs$)?

Nucleoside diphosphate kinase.

Which mechanism does Nucleoside Diphosphate Kinase use to transfer phosphate groups?

Ping-Pong mechanism.

In the first step of the Nucleoside Diphosphate Kinase reaction, where is the phosphoryl group from $ATP$ transferred?

To an active-site Histidine ($His$) residue.

Which enzyme lowers the $ADP$ concentration and replenishes $ATP$ during periods of intense energy demand?

Adenylate kinase.

The force proportional to the difference in electron affinity between chemical species that drives electron flow is the _____.

Electromotive force (emf)

A conjugate redox pair consists of an electron donor and an _____.

Electron acceptor.

Term: Dehydrogenation

Definition: A biochemical oxidation reaction where a compound loses two electrons and two hydrogen ions.

Which class of enzymes typically catalyzes dehydrogenation reactions?

Dehydrogenases.

List the sequence of carbon oxidation states from least oxidized to most oxidized.

Alkane, Alcohol, Aldehyde (Ketone), Carboxylic acid, Carbon dioxide.

What does a positive standard reduction potential ($E^\circ$) indicate about a redox pair's affinity for electrons?

It has a high affinity and tends to take electrons (act as an oxidant).

Electrons tend to flow spontaneously from a half-cell with a _____ reduction potential to one with a _____ reduction potential.

Lower; higher

What is the reference half-reaction used to measure standard reduction potentials?

$$H^+ + e^- \rightarrow \frac{1}{2} H_2$$

Formula: Nernst Equation (at $298 K$)

$$E = E^\circ + \frac{0.026 V}{n} \ln \frac{[\text{electron acceptor}]}{[\text{electron donor}]}$$

What is the mathematical relationship between the change in free energy ($\Delta G$) and the change in reduction potential ($\Delta E$)?

$$\Delta G = -nF\Delta E$$

Standard reduction potentials ($E^\circ$) in biochemistry are conventionally valid only at _____.

Neutral $pH$ ($pH 7$).

Which coenzyme is used specifically as an electron carrier in many biological oxidations, accepting a hydride ion ($:H^-$)?

$NAD^+$ (Nicotinamide adenine dinucleotide).

How does $NADP^+$ differ structurally from $NAD^+$?

It has an additional phosphate group esterified to the $2'$-hydroxyl group of the adenosine ribose.

Reduced $NADH$ and $NADPH$ show a characteristic light absorbance peak at _____ $nm$, which is absent in their oxidized forms.

$340$

How many electrons and protons are accepted by the nicotinamide ring of $NAD^+$ during its reduction to $NADH$?

Two electrons and one proton (as a hydride ion).

Beside electron transfer, $NAD^+$ serves as a source for which group in the bacterial $DNA$ ligase reaction?

The activating $AMP$ group.

Which proteins utilize $NAD^+$ for the deacetylation of Lysine residues to regulate aging and transcription?

Sirtuin proteins.

What is the common vitamin precursor for the synthesis of flavin nucleotides like $FMN$ and $FAD$?

Riboflavin (Vitamin $B_2$).

Unlike $NAD^+$, flavin coenzymes ($FMN$/$FAD$) are usually _____ to their enzymes.

Tightly (or covalently) bound.

How many electrons can the isoalloxazine ring of $FAD$ accept from a reduced substrate?

Either one or two electrons.

What is the name of the stable, one-electron reduced intermediate form of $FAD$?

Semiquinone ($FADH^\bullet$).

Why is the reduction potential of a flavin nucleotide variable rather than fixed?

It depends on the specific flavoprotein with which it is associated.

In the $TCA$ cycle, which intermediate provides energy for $P_i$ addition to $NDP$ to form $NTP$?

Succinyl-$CoA$.

What is the typical standard free energy of hydrolysis ($\Delta G'^\circ$) for $ATP$ in $kJ/mol$?

$-30.5$ (or $-31.8$ as per the provided table).

Concept: High-energy compound

Definition: A chemical compound whose hydrolysis under standard conditions results in a large negative free-energy change ($< -25 kJ/mol$).

How does the removal of $P_i$ by further metabolism affect the free energy of a hydrolysis reaction in a cell?

It drives the reaction forward by decreasing the product concentration, making the actual $\Delta G$ more negative.

Which nucleoside triphosphate is energetically equivalent to $ATP$ and often used in the $TCA$ cycle in animals?

$GTP$.

The standard reduction potential for the $NAD^+/NADH$ half-reaction at $pH 7$ is _____ $V$.

$-0.320$

What structural unit of $NAD^+$ and $NADP^+$ is responsible for accepting and donating electrons?

The nicotinamide ring.

According to the table in the source, which compound has the highest negative $\Delta G'^\circ$ of hydrolysis?

$1,3$-Bisphosphoglycerate ($-52.0 kJ/mol$).

What role does the $\Delta p$ (proton gradient) play in mitochondrial $ATP$ synthesis?

It provides the energy used by $ATP$ synthase to make $ATP$ from $ADP$ and $P_i$.