Unit 2

What does KM refer to?

Concentration of substrate needed to reach ½ of the max

or the affinity of the enzyme for the substrate

what kind of relationship does Km have with enzyme affinity

An inverse relationship

The lower the Km value, the higher the affinity

What does a Vmax refer to?

The maximum velocity of the enzyme catalyzing conversion when its saturated; when all active sites are saturated

What Kcat refer to?

Turnover rate of an enzyme; number of substrate molecules being converted into a product by a single enzyme molecule per unit time when the enzyme is fully saturated

TLDR: catalytic efficiency of enzyme

• kCat = (vmax/[e])

• Higher kcat = more efficient at catalysis

What is the difference between the active site and allosteric site?

Active site - region of the enzyme where the substrate (or competitive/irreversible inhibitor) binds to

Allosteric site - separate site on the enzyme where non-competitive inhibitor can bind to in order to change the conformation of the active site to prevent the substrate binding or being released

How does [E] affect Vmax?

More [E] available = more active sites allows Vmax rate reaction to be higher; vice versa

What is an irreversible inhibition?

Inhibitor is attached to the enzyme’s active site for long periods of time due to covalent bonds

What is the function of penicillin

Penicillin is an antibiotic that is an irreversible inhibitor, it mimics the substrate of glycopeptide transpeptidase (GPT)

• GPT catalyzes the final step in the synthesis of the bacterial peptide can cell walls

• Function: stops bacteria from growing and eventually dies by targeting the cell wall

• Mechanism: penicillin binds to the active site and covalent bonds with a key Ser residue to the bacteria’s active site, resulting in the enzyme being deactivated

What does KM refer to?

when the inhibitor binds to an enzymes active site using weak ionic interactions

How do you calculate Km and Vmax using the lineweaver burk plot?

graph 1/V0 vs 1/s → from this we can calculate:

• y intercept: 1/vmax → go to the line that crosses the y-not, read the number and take the inverse

• X intercept: -1/km → read where the line crossed the x-int, take the minus inverse of that number

What is competitive inhibition

A type of reversible inhibition that occurs when the inhibitor has the same shape as the substrate, competing with the substrate to bind at the active site

• result: only one can bind at the active site at a time, thus catalysis is slowed as there is less ES

What is the effect on competitive inhibition of the Km, Kcat, and Vmax

• vmax: No change (because adding enough substrate can outcompete the inhibitor).

• Kcat: stays the same because catalytic activity is not impacted, only substrate binding

• Km: Increases because more substrate is needed to reach half-maximal velocity (higher KM, the lower the affinity; inverse relationship)

What is the effect on irreversible inhibition of the Km, Kcat, and Vmax

• vmax: lowers [E], thus decreases

• Kcat: changes functional [E], while [E] stays the same thus it will decrease

• Km: does not change; will always have the same affinity

What is non-competitive inhibition

A type of reversible inhibition that binds at the alosteric site (not active site) and inhibits the enzyme

• substrate can still bind but no product will be formed

• Adding more substrate will not outcompete the inhibitor

What is the effect of non-competitive inhibition on a Vmax, Kcat, and Km

• Vmax: decrease because number of active sites available Decreases

• Kcat: if vmax decreases, and [E] stays the same but active site decreases, k at will decrease

• Km: stays the same because enzyme affinity doesn’t change since it is binding at the aloesteric site

What is a protease

A protease breaks down proteins by cleaving peptide bonds by hydrolysis

What is chymotrypsin

A serine protease that is found in the small intestines.

• function: cleaves peptide bonds at the C-terminal after large hydrophobic a.a residues (Tyr, Phe, Trp, Met)

What is the structure of chymotrypsin

Composed of 3 chains (heterotrimer) formed by the cleavage of a single polypeptide

Why is Serine 195 so reactive?

1. Ser 195 can H-bond to the midsole ring on His57

   • result: this positions Ser195 and polarizes the hydroxyl group (has more alkoxide character), turning it into a base catalyst (proton acceptor)

   • It pulls protons from Ser195, making it a better nucleophile

2. Stabilization by Asp 102

   • Asp102 helps to position His57 and stabilize the + charge (that develops during proton extraction), this stabilization makes it easier for His57 to deprotonate Ser195

   • Asp 102 makes His57 more basic, easier to deprotonate Ser195

What amino acids are in the catalytic train

• Asp 102

• His 57

• Ser 195

Ser smith has Asperger’s

What is the first step of the chymotrypsin mechanism?

Substrate binds to chymotrypsin’s active site

• the hydrophobic amino acid residue is recognized by the hydrophobic pocket (S1 pocket)

What is the second step of the chymotrypsin mechanism?

Ser 195 does a nucleophilic attack on the carbonyl chain of the substrate

• result: proton from Ser 195 is transformed to His 57

How does cooperative binding Works in hemoglobin (full tetramer):

When one oxygen molecule binds to one of hemoglobin’s four subunits, it slightly shifts the structure. This weakens the interactions that stabilize the T-state and makes it easier for the other subunits to bind oxygen. With each additional oxygen molecule binding, hemoglobin shifts further toward the R-state, increasing oxygen affinity for the remaining unbound subunits.

What is the difference and one similarity between chymotrypsin and trypsin

• chymotrypsin cuts after large hydropobic groups (Tyr, Trp, Phe, Met)

• Trypsin cuts after Arg or Lys

Both are proteases that have a catalytic triad mechanism for cleaving peptide bonds

How is chymotrypsin/trypsin specific?

The S1 pocket determines specificity

What is the S1 pocket

A deep hydrophobic pocket located in chymotrypsin’s active site where the hydrophobic group of a substrate can bind to

Describe the S1 pocket in the serines proteases

• chymotrypsin: large hydrophobic group

• Trypsin: has a negatively charged Asp at the bottom

• Elastase: has 2 Val in the side chain of the S1 pocket, narrowing it (only small hydrophobic residues can fit)

what are the 4 protease groups

1. serine protease family

   • ex. thrombin (blood clotting enzyme)

2. cysteine proteases

   • use cysteine side chain as a nucleophile

3. aspartyl proteases

   • use Asp to make H2O a strong nucleophile

   • ex. HIV protease

4. metallaprotease

   • use a metal ion and H2O as a nucleophile

cut off at hemoglobin

what is metabolism

a highly integrated network of chemical pathways that extract energy from the environment to make energy

what are the 2 types of metabolism

1. catabolism - reactions that use building block molecules into useful energy

   • cat = all of benji’s small acts makes me love him more

2. anabolism - reactions that use energy to break down complex structures into simple ones

what must happen for a pathway to proceed to its final product

1. the individual reactions must be specific for one set of products

2. all reactions must be thermodynamically favored (negative delta G)

what are the conditions at the standard free energy change

• T = 298 K (25 degrees)

• pressure = 1 atm

• pH = 7

• all [ ] = 1M

what is the most common way to drive cellular endergonic reactions?

couple them to adenosine triphosphate (ATP) hydrolysis

• this makes it so the equilibrium shifts to favor the products

what is the structure of adenosine and ATP

• adenosine = adenine + ribose (nitrogenous base)

• adenosine triphosphate (ATP)

  • adenine (nitrogenous base)

  • ribose: a 5-carbon sugar that connects to the phosphate group using phosphodiester bonds

  • 3 phosphate groups: linked by high-energy phosphoanhydride bonds

-

where are the phosphodiester bonds in ATP

These bonds connect the phosphate group to the ribose sugar in ATP (specifically between the alpha phosphate and the 5' carbon of ribose).

These bonds are not considered high-energy like the phosphoanhydride bonds.

why is there so much energy in the phosphodiester bonds of ATP

1. relief of charge repulsion: the 3 phosphate groups are negatively charged and repel each other. Holding these charges so close together takes a lot of energy, so breaking the bonds relieves this repulsion.

2. resonance stabilization of pi: the phosphate group is more stable by itself than when it is attached to ATP

3. ionization of ADP: once ATP is hydrolyzed the products (ADP and Pi) can be more easily stabilized by water molecules through hydration

4. increase in entropy: the hydrolysis of ATP increases entropy, which is thermodynamically favored

what is phosphoryl transfer

the movement of a phosphate group from one molecule to another molecule

what is phosphoryl transfer potential

• the likelihood a molecule can transfer a phosphate based on their ΔG° of hydrolysis of the phosphate

what does a more negative ΔG° mean on the PTP

• the more negative the ΔG° = the higher the PTP = really wants to give away the phosphate group

  • ex. the higher the PTP, the easier it is to phosphorylate something with a lower PTP

what is the ΔG° of ATP synthesis

^+30.5

what is the reaction mechanism for the creation of ATP using creatine phosphate

creatine phosphate + ADP ←→ ATP + creatine

what is creatine phosphate

• it is used to generate ATP in the muscles during the first few seconds of heavy muscle contraction when [ATP] is low

  • it bridges the gap until the catabolic pathway kicks in

what is the difference between an L or D sugar

• D sugar = OH is on the right

• L sugar = OH is on the left

how can you switch from a D to L sugar

you need to flip everything

what is an epimer

type of diastereomer that differ only at one chiral carbon

what is the difference between a ketose and aldose

• ketose = contains a ketone group

• aldose = contains an aldehyde group

what is this?

an epimer

• D-glucose & D-galactose

• D-glucose & D-mannose

what is an anomer?

• when a carbon cyclizes, the new chiral carbon formed is the anomeric carbon

• anomer = chirality = has 2 forms

what is the difference between a beta and an alpha anomer

• alpha anomer = when the OH is below

  • alphas are trans

• beta anomer = when the OH is above the ring

what is mutarotation

• the conversion between alpha and beta anomers of a carbohydrate in aqueous solution, resulting in a change in the optical rotation of the solution.

  • it is a spontaneous process in glucose and fructose

why does mutarotation occur

Mutarotation occurs because the α and β forms are not stable alone and constantly interconvert through the open-chain form.

what is glycosylation

the process of attaching carbohydrate groups (glycans) to proteins, lipids, or other organic molecules.

what is a glycosidic bond

• when a cylic sugar reacts with a glucose/fructose’s hydroxyl or amine group to form a glycosidic bond

• A glycosidic bond is a covalent bond that connects a carbohydrate (sugar molecule) to another molecule,

how do you draw glucose

1. draw a 6-membered ring

2. an O replaces the top right C

3. alternate between H and OH

4. at the end draw the CH2OH

what are the monomers that make up sucrose

• glucose (alpha → beta) fructose

what are the monomers that make up lactose

• galactose (B1 → C4) glucose

What is the first step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: glucose is phosphorylated using ATP by hexokinase on C6, turning into glucose-6-phosphate

• enzyme: hexokinase

• why do we do it? phosphorylation of glucose traps the glucose in the cell (negatively charged, cannot cross the membrane)

what step is this?

#1: phosphorylation of glucose to glucose-6-phosphate by hexokinase

What is the second step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: glucose-6-phosphate is converted into fructose-6-phosphate by phosphoglucoseisomerase

  1. converts glucose-6-phosphate into an oen chain

  2. repositions the double bond, forming fructose-6-phosphate (open chain)

  3. cycalize to form furanose

• enzyme: phosphoglucoseisomerase

• why do we do it? fructose is more symmetrical than glucose

what step is this?

#2: isomerization of glucose-6-phosphate into fructose-6-phosphate

What is the third step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: transfer of phosphate from onto onto fructose-6-phosphate, converting it into fructose-1,6-bisphosphate by phosphofructokinase

• enzyme: phosphofructokinase

• why do we do it? To make fructose even more symmetrical since there is only one phosphate (on one side)

what step is this?

#3: phosphorylation of fructose-6-phosphate into fructose-1,6-bisphosphate by phosphofructokinase

What is the fourth step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: fructose-1,6-bisphosphate is cleaved into dihydroxyacetonephosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) by aldose

• enzyme: aldose

• why do we do it? Splitting glucose makes glycolysis more efficient at capturing energy

what carbons is DHAP and G3P composed of?

• from the original fructose-1,6-bisphosphate…

  • DHAP = carbons 1-3

  • G3P = carbons 4-6

what is this step?

#4: cleavage of fructose-1,6-bisphosphate into GAP and DHAP

What is the fifth step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: isomerization of dihydroxyacetonephosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) by triose phosphate isomerase

• enzyme: triose phosphate isomerase

• why do we do it? GAP is more favorable than DHAP

what is this step?

#5: isomerization of dihydroxyacetoe-phosphate into glyceraldehyde-3-phosphate by triose phosphate isomerase

What is the 6th step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: phosphorylation and oxidation of GAP to 1,3-Bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase

  1. Formation of a Hemithioacetal Intermediate:

     • GAP (glyceraldehyde 3-phosphate) reacts with a cysteine residue in the active site of GAPDH (enzyme)

     • The thiol group (–SH) of cysteine nucleophilically attacks the carbonyl carbon of GAP, forming a hemithioacetal intermediate.

  2. Oxidation of the Hemithioacetal to a Thioester:

     • NAD+, also bound in the active site, accepts 2 electrons and 1 proton from the hemithioacetal intermediate.

     • This oxidation converts the hemithioacetal to a thioester intermediate, while NAD+ is reduced to NADH.

     • The thioester bond is high-energy, facilitating the next step.

  3. Release of NADH and Replacement with NAD+:

     • The newly formed NADH leaves the active site and is replaced by a fresh NAD+.

     • This exchange is crucial because the presence of NAD+ activates the enzyme for the next catalytic cycle.

  4. Orthophosphate Attack and Product Release:

     • Inorganic phosphate (Pi) attacks the thioester carbonyl, forming 1,3-bisphosphoglycerate (1,3-BPG).

     • The thioester bond is cleaved, releasing the product and regenerating the free cysteine residue in the enzyme’s active site.

• enzyme: glyceraldehyde-3-phosphate dehydrogenase

• why do we do it?

  • Produces NADH, which carries electrons to the electron transport chain (ETC).

  • Forms 1,3-BPG, a high-energy intermediate that will participate in substrate-level phosphorylation in Step 7 to produce ATP.

  • This reaction is exergonic (releases energy), which helps drive the subsequent ATP formation.

what is this step?

#6: oxidation and phosphorylation of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase

What is the 7th step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: transfer of the phosphate from 1,3-BPG onto ADP to make ATP and 3-phosphoglycerate

• enzyme: phosphoglycerate kinase

• why do we do this step?

  • first time we form ATP in glycolysis

  • 1,3-BPG is a high-energy intermediate with a high phosphoryl transfer potential.

  • Converting it to 3-phosphoglycerate (3PG) while generating ATP ensures that the energy stored in the high-energy bond is not wasted.

what step is this?

#7: ATP generation via substrate-level phosphorylaton from 1,3-biphosphoglycerate to ADP by phosphoglycerate kinase

What is the 8th step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism:

  1. Phosphate Group Shuffle:

     • The enzyme phosphoglycerate mutase shifts the phosphate group 3-phosphoglycerate to 2-phosphoglycerate (2PG)

     • This involves a temporary phosphorylation of the enzyme itself by a histidine residue, forming a phospho-enzyme intermediate.

  2. Removal of phosphate

     • the phosphate group comes off the intermediate (2,3-BPG) and re-phosphorylates the enzyme

• enzyme: phosphoglycerate mutase

• why do we do it?

  • The rearrangement creates 2-phosphoglycerate, which is a better substrate for dehydration in the next step.

what step is this?

#8: intramolecular shift of 3-phosphoglycerate into 2-phosphoglycerate by phosphoglycerate mutase

What is the 9th step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: enolase removes water from 2PG by enolase, forming phosphoenolpyruvate

• enzyme: enolase

• why do we do it?

  • enol is more unstable than keto, meaning it has a higher PTP, which will help us generate ATP in the next step

what step is this?

#9: dehydration of 2-phosphoglycerate, forming phosphoenolpyruvate by enolase

What is the 10th step of glycolysis, what enzymes mediate it, and why do we do it?

• mechanism: pyruvate kinase transfers a phosphate group from phosphoenolpyruvate to ADP, forming ATP and pyruvate

  • phosphoenolpyruvate immediately goes to its enol form (pyruvate) once the phosphate comes off

• enzyme: pyruvate kinase

• why do we do it?

  • forms pyruvate which is needed in the citric acid cycle

what step is this?

#10: generation of ATP and pyruvate via substrate level phosphorylation from phosphoenolpyruvate by pyruvate kinase

what is decarboxylase and carboxylase

• decarboxylase = removes carboxyl

• carboxylase = adds carboxyl group