Biochemistry Exam 2

A substrate with negative charges interact with what amino acids?

Positively Charged, basic

How is the affinity of an interaction determined?

Strength and stability of binding interactions

• higher kd = less affinity

• higher ka = greater affinity for substrate to protein = more binding

What is being measured in the affinity of an interaction?

The measurement assesses the strength and stability of binding interactions between a substrate and a protein.

How are the ka and kd defined?

ka is the association constant, and kd is the dissociation constant.

What is cooperativity? What kind of graphing curve does it have?

binding produces affinity for binding at another site

• sigmoidal curve

What drives cooperative binding of O2 in hemoglobin?  What are the T and R states of hemoglobin? How do they contribute to cooperativity?  Why is cooperative binding of O2 important for Hemoglobin’s function in O2 transport from the lungs to peripheral tissues?

• as O2 binds to the sites, the affinity grows in hte other sites

• the sites move from T state (tense, low affinity) to R state (relaxed, high affinity) —> gradual relaxation

• theres a lot of oxygen in hte lungs whil puts it in a more relaxed state moving through the curve; peripheral tissues get more relaxed as oxygen reaches them

What is allostery? What is the relationship between cooperative binding and allostery?

the binding of one site affects another

• cooperative binding is a more specifc verson of allostery where it increases affinity as in binds

How can ATP hydrolysis be used to drive protein conformational changes?

• Ex. muscle contraction : as ATP hydrolyzes, myosin and actin are aligned in the right place to contract then return to normal positioning

How can protein conformational changes be harnessed to provide information in biological processes such as growth factor signaling?

acting as molecular switches

• ligand bindings triggers structural shits that alter enzyme activity (allosteric changes) facilitating signal transduction by altering protein affinity

1. would happen to contraction if ATP hydrolysis was blocked, but ATP binding was not?

2. Why are ADP and Pi release equally as important for muscle contraction as ATP hydrolysis?

3. What would happen if Pi release was blocked

1. failure to contract

2. drive “power stroke” or conformational change that align myosin and actin. and cross-brige attachment of myosin from actin which restarts the cycle

3. If Pi release blocked, no power stroke or conformational change

How do enzymes increase the rate of catalysis? What is their effect on ΔG?

• Overcome energy barriers

• Affects rates, not DG’0 or DG

• Binding energy contribution to account for first law of thermo

Chymotrypsin mechanism****

How does affinity to substrate affect the reaction?  What is the induced fit model?

faster when affinity is higher

• induced fit is when an enzyme changes conformation to fit the substrate (transition state), pushing transformation forward

 1. Why would an enzyme with high complementarity to the substrate be a poor catalyst? 

2. What structure should an efficient enzyme prefer?  What is the “transition state”?

3. What are transition state analogs?How does binding energy affect enzyme function? How does binding energy contribute to catalysis?

1. Higher complementarity would not push the substrate to the product (not fitting the transition state)

2. the transition state (in between the substrate and the product

3. they mimic the transition state of the substrate. Binding energy is released whebn the substrate binds and helps overcome activation energy. It stabilizes the transition state (pushed to product).

How are enzyme kinetics measured/determined—how is data collected to create a Vo/[S] graph? What parameter is held constant for these graphs? What is the Vo of a reaction? A Vmax? What is Km? How are they determined?

• What would two enzyme reaction graphs look like for the same enzyme and substrate, but with twice the amount of enzyme added in one of the reactions? 

 What is the relationship between Km and affinity of enzymes for substrates? Does Km only describe affinity of the enzyme for the substrate?

What is kcat/Km? How is this related to enzyme efficiency?  What does it mean that two enzymes can have the same Km for a substrate but different efficiencies? How can this occur?

Double-Reciporacal Plot

• line

• Lineweaver-Burk Plots for inhibition

• competitive vs Uncompetitive vs. Noncompetitive

Competitive inhibition Kinetics

• km increases (more substrate required to compete with the inhibitor) — x intercept chages, steeper slope

• Vmax unaffected — y intercept does not change, once out compete, get to max velocity just need more substrate— turnover didn’t change, enzyme can still work at its max rate

• competitor bind to the ENZYME, EI complex, not ES complex

• doesn’t always bind to the active site it can be an allosteric

  inhibitor: its binding to an

  allosteric site could prevent the

  enzyme from binding substrate.

  If the substrate binds first, the

  inhibitor can’t bind and catalysis

  can happen. The “competition”

  is for binding the enzyme first,

  independent of where that

  binding occurs

noncompetitive inhibition

• bind at allosteric regulatory site (at E+S, beginning of reaction)— decreases activity of enzyme/change access to active site

• Km unaffected (can’t increase substrate to overcome allosteric inhibition) — x-intercept stays the same

• Vmax reduced — y intercept changes, Inhibitor can bind to enzyme with or without substrate, so apparent Km doesn’t

  change but Vmax decreases

• mixed inhibition: could bind to E+S or ES complex (2 options)

• Both S and I can be bound to enzyme at same time or separately; i.e., Substrate binding is

  unaffected, but catalysis is decreased

• Inhibitor binds equally well to E or ES,

  but it’s binding affects the conformation

  of the catalytic site, decreasing the rate

  of catalysis

uncompetitive inhibitor

binds enzyme-substrate complex (after they come together), then controls and stops turnover/process

• Km reduced - saturating protein earlier, can’t release substrate (temporarily locked → bad = taking enzyme out of solution

  • affinity is increasing because adding ESi complex→ favors backward reaction→ foming more ES (Le Chatlier’s)→ need lower Km for substrate to bind to

• Vmax reduced

• change proportionally to one another

• Inhibitor will only bind ES complex; prevents or slows

  ES→ EP and ES→E+S; Vmax is lower but apparent Km is

  also lower as product release is slowed and ESI decreases

  effective enzyme concentration,

• Uncompetitive inhibition cannot be

  reversed by increasing [S]

What is Km? What would it mean if the Km is low?

affinity for substrate

• if lower, has high affinity because not a lot of substrate is required to reach Vmax

• half of Vmax

***Vmax = y intercept

What is the Vmax?

the max velocity of an enzyme reaction/ max turnover

What does the inhibitor bind to in uncompetitive inhibition?

ES complex at an

What does a high Km mean?

low affinity

• you would need to throw higher concentration of substrate at the enzyme because the enzyme doesn’t want it as bad (it’s lazy)

In a hydropathy index, the higher or more postive the index, means the residue is_______

hydrophobic

Irreversible Inhibition

• totally kills the enzyme, the reaction doesn’t return - ***typically because of a covalent bond between inhibitor and enzyme → others usually release making them reversible

Enzyme Regulation

• post-translational modification

  • phosphorylation - glucogenesis, kinase activity (opposite is phosphotase activity)

  • ubiquination - get rid of protein after doing its job

  • ex. acetylation, adenylation,

****pathways

in allosteric regulation, is it exactly like an on or off switch?

no, there is always leaky transcription, never truelyl one or off, just different levels of activity

• to not run out of resources or burn out the system

***why drugs are not always effective — also apply to competitive inhibition

Regulatory enzymes

• pathway control

• often composed of regulatory subunits and catalystic subunits

• exhibit non-Michaelis-menten kinetics

• some exhibit cooperativity with small increases in the substrate

Where is the activity modulation binding site/ Allosteric Site?

Typically in regulatory subunit physically distant from the catalytic site in the quaternary strcucture

Homotropic Regulation

one of the substrates acts as an allosteric regulator

Importance of first phosphate in glycolysis?

to prevent reversibility of the process

• added by PFK

• phosphate adds to active site, ATP binds to active (high affinity) and allosteric site (low affinity, only binds when theres a lot of ATP→ negative regulation -→ homotropic regulation

phosphate→ glucose→ fructose→

Heterotropic Regulation

• regulatory enzymes regulated by negative and positive modulators

homotropic and heterotropic working together to make a “dial” that turns the system up or down, not on or off

Aspartate Transcarbomoylase (ATCase) and heterotrophic modulation

• important regulatory enzyme in the pathway of pyrimidine biosynthesis (make nitrogenous bases/nucleotides)

• formation: carbamoyl Phosphate + aspartate

• has 12 polypeptides assembled into 6 catalytic units plus 6 regulatory subunits

• CTP binds to it in inactive T state → releases in active R state

• Homotropcic Regulation: increases in aspartate and carbamoyl-P cause gradual T→ R state change

• Heterotrophic regulation: moving enzyme in two different directions depending on the needs of the cell

  • Postive Heterotrpic modulation: ATP stimulates it → lots of excess energy → anabolic processes (replicate DNA, make more nucleotides)

  • Negative Modulation: CTP binding to allosteric sites

What is CTP?

pyrimidine nucleotide

• inhibits ATCase in negative modulation

Why does it make sense for CTP to negatively modulate ATCase? (pyrimidine biosynthesis)

• in the subunits there are multiple places for CTP to bind, and when it binds at these allosteric sites, it inhibits through negative modulation

Why would high levels of ATP be a logical activator of ATCase?

Postive Heterotrpic modulation (ligand binds to allosteric site increasing affinity): ATP stimulates it → lots of excess energy → anabolic processes (replicate DNA, make more nucleotides)

Homotropic activation in ATCase

an increase in either substrate promotes shift from T→ R state yielding sigmoidal kinetics

• similar to cooperative binding, the sigmoidal kinetics is due to the combination of two distinct kinetics as a function of [S]

Heterotrophic Modulation

• ATP positively modulates and promotes R state even at lower [S]

• CTP negatively modulates and promotes T state even at high [S]

Regulatory enzymes provide pathway _______ ________ — a common theme in metabolic regulation

“feedback inhibition”

ex. Isoleucine biosynthesis pathway regulation

• Threonine substrate→ isoleucine allosteric regulation

In a sigmoidal curve, shifting to the left means the enzyme what?

has more affinity, is more effective at binding substrate and creating product

Isoleucine Bisynthesis regulation pathway

Glycogen phosphorylase

• Glycogen: Stored form of glucose kept largely in muscle and liver. Mobilized when here is an urgent need for glucose oxidation or during low nutrition.

• Glucose is freed as Glc-1-P by a phosphorylysis reaction (addition of phosphate and breaking of glycogen)--- it is then converted to

  Glc-6-P for entry into glycolysis when other sources of glucose are low

Explain how glycogen phosphorylase exists as an equilibrium of two states

(Relaxed) R-State: More active state

(Tense) T-State: Less active state (still active tho)

• Phosphorylation of Serine 14 (A in diagram)by Phosphorylase Kinase shifts the equilibrium so the more active R state is favored (still possible to be inactive but the active state predominates)

• Hormone signaling, such as adrenaline,

activates Phosphorylase Kinase

• further modulation of sigmoidal curve: some heterotropic and homotropic regulation: in A more glucose-6-P → T state, in B more glucose or ATP → T state

What is adrenaline, what physiological

response does it direct, and why would

adrenaline’s activation of phosphorylase

kinase make sense in muscle?

Covalent and/or Allosteric Regulation of Glycogen Phosphorylase

Conversion of T (less active) to R (more

active state) is also enhanced by AMP

• enhanced by AMP + phosphoylation

What does increased [AMP] in the cell indicate

and why would increased Glycogen

phosphorylase activity be a good response in

muscle cells?

increased AMP → PKA activity→ phosphorylase kinase phosphorylated → activated phosphorylase A→ mobilized glycogen storage

• activated when there isn’t enough glucose : starving

Glycogen Phosphorylase is highly regulated by Both Kinetic and Post-Translational modifications (PTM) Controls: ***pathway****

• Phosphorylation on Serine

  converts it from a lower

  activity form

  (Phosphorylase b) to the

  higher activity form

  (Phosphorylase a)

****Note roles of metabolic hormones,

Insulin (high blood Glc) and

Glucagon (low blood Glc) and how

they influence the activity of the

enzyme and determine whether or

not to release Glucose stores…...

• insulin (removing phosphate groups): make more glycogen, absorbed sugar, anabolic activity, phosphorylase activity deactivated phosphorylase and brings it back to phosphorylase B

• glucagon: increase phosphotase activity

• when out of whack -→ diabetes

*****Think about the goals of insulin and glucagon signaling and why their opposing regulation of glycogen

phosphorylase makes sense….

Double- Reciprocal Plot (Lineweaver-Burke Plot)

This way of visualizing the kinetic data for an enzyme

reaction yields information on the mechanisms of the

reactions, but is most often used to determine the

mechanism of an inhibitor to the reaction

aKm

aparrant Km of the substrate in hte presence of an inhibitor

Does non-competitive have a sigmoidal curve?

yes

Does Uncompetative inhibition have a sigmoidal ccurve?

no, not allosterically regulated but binds to the ES complex

Regulatory enzymes exhibit reaction kinetics that are _______ due to changes in kinetics that occur with additional substrate binding (cooperativity)

sigmoidal

To survive cold environments, fish would need more _____ in their membranes

unsaturations

Flippases

• send lipidsagainst concentration into cell using ATP

Floppases

send lipids against concentration gradient outside of cell using ATP

ER Scramblase

• move lipids down gradient

• restore equilibrium/maintain balance if there is asymmtry where there shouldn’t be

cell and organelle membrane advantages

structure, permeability keeping things in or out, doing work (electron transport, receptors for signaling, cellular communication and identification)- outweigh disadvantages

Which has more asymmetry Plasma Membrane or ER and why

• more asymmetry in plasma membrane than ER

  • ER doesn’t need asymmetry so there is no flippases or floppases present

Types of Flippases

• require ATP: flippases and floppases

• Don’t: scrablases

**both are enzymes

Can you establish a concentration gradient without ATP?

no

Passive vs. Facilitated diffusion kinetics

facilitated (similar to non-regulatory curve/ligand receptor binding) - increase in movement, but get saturated and level off

passive - totally dependent on concentration gradient

Integral membrane proteins embedded within lipid bilayer

alpha-helical structure and beta-barrel structure

• eukaryotes have channels made of alphahelicies

• beta-barral in prokaryotic organisms: ex.pathogens make channels

What goes easy through the membrane

small, nonpolar

What have trouble and what can’t go through membrane

big, polar - trouble

ions- can’t

Types of membrane transport

active (require energy/ATP, postive DG - moving hard to move molecules, unidirectional) and passive (diffusion and facilitated diffusion, negative DG, reversable)

difference between uniport, cotransport symport and antiport

*****can be facilitative or active

Facilitated transport

• down concentration gradient/across energy barrier -→ eliminates need to remove hydrogen bonding/hydration shell (would be required with simple diffusion)

• hydrophilic molecules require transporter

• hydrophobic molecules don’t need it

• transporters can have conformational change (even though don’t need ATP — stacked cylinders rotating in or out to be specific channels

  • alpha helicies - these can rotate: open or close → more specificity/regulated

  • beta-barrel structures have no control

What types of transporter is glucose transporter?

facilitated transporter

• induced fit → conformational change→ product release

• T1 = ES

• T2 = EP

• not reversible

• what would make it reversable?

Aquaporin

• alphahelical structues

• function: maintain fluid balance in many tissues, urine concentration, tears, edema etc.

Na+'/Ka+ Pump / Na+K+ ATPases

• active transport: energy depended to move against gradient

• antiport

• makes cytosol more negative than outside: 2K+ in 3Na+ out, ECF or blood plasma: positve

• simultaneously pump Sodium and Potassium

  against their concentration/ electrochemical

  gradients

  Results in charge imbalance across a

  membrane creating membrane potential for

  current….action potentials in neurons

• P-type ion pump: reversible autophosphorylation of the protein

Ca2+ ATPase

• type of transport: active

• regulate intracellular calcium levels

When there is no ATP, what can make ATP?

electron transport chain

Is glycolyis anerobic?

up to the production of pyruvate yes, but then it requries oxygen to the ETC

In glycolysis, lots of NADH does what?

push the reverse reaction (regulating metabolic “flux” overlaping with their roles as electron carriers for RedOx chemistry: high NADH drives reduction

chemistry; high NAD+ drives oxidation chemistry. High NADH also slows glycolysis–

reduction of pyruvate to lactate helps alleviate this “block” in exercising muscle

Glycolysis

glucose (6 carbons) → 2 pyruvate (3 carbons): oxidation

• energetically favorable overall, but energy expenditure in Preparatory phase

  is required to provide Payoff in the energy-producing phase

• highly favorable reactions are sites of regulation– to control the pathway and

  its direction, the process must be blocked before each plunge off the “cliffs”

more from summary of lecture 10….

• afterwards, Citric acid cycle creates ATP with ETC

• glucose into cell, add phosphate (anchored in cell), make into fructose with P, splits into two 3 carbond moelcules, 2 steps into PEP, into 2 pyruvate

  • uses ATP in steps 1 and 3, oxydized by NAD+ in step 4, creating ATP in steps 5 and 7

  • ATP needed to phoshporylate

sugars on the outside of cells are for

communication, structure, recognition and identification

• glucose anabolism to cell wall and extracellular matrix to create structural polymers

Energetics of glycolysis

• more endergonic in standard conditions than exergonic

  • in nature, bring down energy required to keep them going with enzymes, energy coupling, compartmentalization

  • Becoming more favorable inside the cell - key steps required big changes in free energy

• hydrolyzing ATP has big energy changes (investing into phosphoylation with Hexokinase (keep glucose inside cell) and Phosphofructokinase (commit moleculeto glycolysis))

• regulate the big drops by facilitating for blocking it —these places are difficult to reverse because going down the hill is easy, going up is hard

Highly favorable steps of glycolysis

a) sites of regulation

b) reactions that Gluconeogenesis (reverse

pathway) has to avoid or go around

• catalyzed by hexokinase, phosphofructokinase-1 (PFK1), and pyruvate kinase.

2 phases of glycolysis

preparatory phase (glucose → G3P) - Sets up glucose as a higher energy substance prepped for -lysis and eventual extraction of energy: An up front investment of 2 ATP

payoff phase (G3P → Pyruvate)- Rearranges carbons to

form strong phosphate donors to

phosphorylate ADP; Reap a 100%

return on ATP investment, with some

NADH as a bonus

in ETC, what is NADH? what is made?

electron donor to make ATP

What can feed into glycolysis?

• many sugars

  • lactose (made of galactose and glucose) - hard because the galactose need extra steps and enters glycolysis in a different part than glucose

  • sucrose - easy

In lactose, what is fed in where? Why is it not favored for glycolysis?

glucose → glycolysis

galactose → broken down into UDP-gal→UDP-glu→ glucose 1-phosphtate (not favored for glycolysis because of extra steps, extra time and energy investment

What is the regulatory enzyme in glycolysis?

PFK-1

When is energy required in glycolysis?

• phosphorylate to keep in cell

• destabilize to split into two molecules

What is the difference between fructose 1,6-Bisphoshate and fructose 2,6-Bisphosphate?

fructose 1,6-Bisphoshate : component in glycolysis in the preparatory phase

fructose 2,6 - Bisphosphate - stimulates glycolysis by activating PFK 1 and inhibits gluconeogenesis

2 important enymes in step 1 of glycolysis

Hexokinase and Glucokinase

Hexokinase/Glucokinase Reaction

1. traps glucose in the cell → glucose-6-phosphate

2. raises glucose core energy level for lysis reactions

• changes glucose to something different so that the glucose transporter reaction is no longer reversible

What can Glucose- 6- phosphate be turned into? ***metabolic cross road

• Glucose-1-phosphate (on different carbon) → glycogen (energy storage)

• 6-phospoglucono-delta?-lactone → ribose-5-phosphate→ nucleotide biosynthesis

• fructose-6-phosphate→ glycolysis (oxidation of fuel)

2 isoforms of hexokinase

• Hexokinase I-III

  • low Km

  • where low levels of sugar are because of high affinity

• Glucokinase (Hexokinase IV)

  • higher Km

  • sigmoidal shape - allostery (affinity changes)

  • primarily in liver, a tissue where glucose storage occurs

  • metabolic sensor

  • important for long-term homeostasis - required sustained insulin release

What is ATP to PFK I?

negative allosteric inhibitor

The key components in going from Fructose 6-phosphate-→ fructose 1,6-bisphosphate

What acts as the opposite of PFK-1in glycolysis, for gluconeogenesis?

FBPase-1 (removes 1 phosphate with H2O)

What inhibits FBPase-1 in gluconeogenesis?

AMP

What is gluconeogenesis?

synthesis of glucose from pyruvate

Where does the first step of oxidization in glycolysis occur?

beginning of payoff phase to create 1,3-BPG

What does Adolase yield in glycolysis?

2 molecules of G3P

GAPDH– Glyceraldehyde-3-P Dehydrogenase

G3P → 1,3-bisphosphoglycerate

Two Step Reaction:

1. Highly exergonic oxidation of aldehyde

to carboxylic acid coupled to Reduction

of NAD+

2. Highly endergonic transfer of inorganic

phosphate to yield high energy

compound 1,3-BPG

3. NOTE: Reaction requires oxidized form

of NAD

Although the sum of the DG of

The reaction is somewhat

unfavorable, the next reaction

quickly removes 1,3-BPG and

thus decreases Q and lowers

overall DG

PEP is a high energy ______ donor

phosphate