Exam 4 Flashcards
Metabolic pathways are constructed from several different individual chemical reactions. For a metabolic pathway to occur, it must satisfy two criteria:
- the individual reactions must be specific
- convert a specific substrate to a specific product
- the enite set of individual reactions that make up a pathway must have an overall negative free energy change (deltaG)
FAD
- flavin adenine dinucleotides
- the second electron relay molecule in fuel oxidation
- oxidized = FAD
- reduced = FADH2
- can accept two electron and two hydrogens
What does KM value describe?
- describes the affinity of an enzyme for a substrate
- High = weak substrate binding
- Low = strong substrate binding
- for most enzymes, value ranges from 10-1 to 10-7M
Addition or removal of functional groups
addition of functional groups to double bonds or their removal to form double bonds
NADH
- nicotinamide adenine diculeotide (NAD)
- major carrier of electrons in the oxidation of fuels
- during oxidation, oxidized NAD+ accepts a hydride (hydrogen plus two electrions) to form NADH
Define Uncompetitive Inhibition
- an inhibitor that binds to the enzyme only after the substrate is bound
- binding site for inhibitor is formed only when substrate is bound
- inhibitor binds ot a site other than the active site
- cannot overcom uncompetitibe inhibition simply by adding more substrate
- bind only to ES complex to form ESI
- ESI complex cannot form product resulting in lower Vmax
- lowers KM
- b/c it depletes ES
- to maintain equilibrium, E binds more S at lower concentrations
Substrate A can be broken down to products B and C with an overall free energy change of +21 kJ/mol. Substrate B can then be broken down to product D with an overall free energy change of -34 kJ/mol. What is the overall free energy change from A being converte to products C and D.
The overall reaction of A being converted to products C and D has a thermodynamically favorable free energy change of -13 kJ/mol
What is Feedback Inhibition?
- a cellular control mechanism in which an enzyme that catalyzes the production of a particular substance in the cell is suppressed when that substance has accummulated to a certian level thus balancing the amount provided with the amount needed
What is Catalytic Efficiency?
- kcat / KM
- an enzyme that binds substrate tightly and rapidly turns substrate to product is a highly efficient enzyme
- an ensyme that binds substrate loosely and slowly turns substrate to product is not an efficient enzyme
What is Steady-State Assumption?
- once a reaction has started, the concentration of ES remains constant
- ES is being converted to product
- ES is converting back to free enzyme and substrate
- allows for the derivation of the Michaelis-Menten equation (pg 231-232)
What are the two classes of Multiple Substrate Reactions?
Sequential
Double-Displacement
reactions involving two substrates and two products
What is Metabolism?
- series of chemical reactions that begin with a particular molecule and convert it into some other molecule or molecules in a carefully designed and highly regulated fashion
- refers to the complex network of how molecules are both synthesized and degraded
Activated carriers of electrons for fuel oxidation
- these molecules relay electron during fuel oxidation
- molecules are pyridine nucleotides and flavins
True or False
The more reduced a carbon compound is, the smaller its potential to generate free energy via oxidation
False
The more reduced (more energy) a carbon compound is, the greater its potential to generate free energy via oxidation
Define Rate Constant (k)
- describes velocity
- states how fast something progresses
- rate constant k is directly related to the concentration of A
V = k[A]
Define Affinity-Label Inhibitors
- a type of irreversible inhibitor
- inhibitors that are structurally similar to the natural substrate and covalently bind to the active site
Why do we measure the rate (V0) at very early times near time zero in kinetics?
- looking at only the forward reaction (substrate to product) and not the reverse reaction (product back to substrate)
- measure early when the rate is at its highest prior to reaching equilibrium
- often times the product can inhibit the enzyme (product inhibition) which slows the rate
What is the Citric Acid Cycle?
- one of the 2 pathways that acetyl is shuttled through in Stage 3
- the acetyl group is oxiized to CO2
Activated carriers of electrons for fuel oxidation
- these molecules receive electrons during oxidation and relay them on to other molecules (oxygen) to make ATP
Define Kinetics
- study of the rates of chemical reactions
What are the promary sources of ATP production?
oxidation of molecules such as glucose and lipids via catabolism
What are the two types of Metabolism?
Catabolism
Anabolism
There is a equation that was used prior to the Michaelis-Menten Equation. Write out that equation.
- Lineweaver-Burk (Double Reciprocal) Plot
- gives a straight line
Fill in Blank
As higher amounts of product is formed per time (V) is _________ as higher concentrations of substate are added
increasing
Ligation requiring ATP clevage
formation of covalent bonds
carbon carbon bonds
If you measure V0 at different substrate concentrations you get a graph. Draw this graph with appropiate units.
- Rate (V0) will increase until it levels off at high substrate concentrations
- all enzyme has been saturated with substrate
Explain the 3 stages of energy production
Stage 1
- fuel molecules such as fats, sugars, and proteins are digested and broken down into smaller components
- small components are absorbed by the intestine and transported to cells
- no ATP is produced at this stage
Stage 2
- small molecules are further converted to a few simple compounds, the most common being a two carbon acetyl group linked to coenzyme A
- a small amount of ATP is produced during this process
Stage 3
- ATP is generated from the comlete oxidation of Acetyl CoA
- Acetyl is shuttled through two pathways
- Citric Acid (Krebs, or TCA)
- Oxidative Phosphorylation
- Acetyl is shuttled through two pathways
What is the equation that Rate decribes?
V= -deltaA/deltaT
(negative due to it being a forward reaction; positive if it’s a reverse reaction)
where A is products and T is time
as A decreases P will increase so…
V= deltaP/deltaT
What does a high KM and a low kcat describe?
An enzyme that binds substrate loosely and slowly turns substrate to product is not an efficienct enzyme
True or False
A reaction can only proceed (is spontaneous) if it has an overall positive free energy change
False
A reaction is spontaneous if it has an overall negative free energy change
What kind of reaction is the following?
Aspartate Aminotransferase
- double displacement reaction
- transfers an amino group from aspartate to alpha-ketoglutarate
- oxaloacetate must release from the enzyme before alpha-ketoglutarate can bind
Activated carriers of two-carbon fragments
- activated and transport molecules needed to make ATP
Define Noncompetitive Inhibition
- a type of reversible inhibition
- when an inhibitor ans substrate can bind the enzyme simultaneously
- substrate and inhibitor bind to two different sites on the ensyme
- differs from uncompetive inhibition in that the inhibitor can bind both free enzyme and ES complex
- cannot be overcome by adding more substrate
- lowers Vmax
- does not alter KM
Define Irreversible Inhibitors
- molecules that form covalent bonds with the enzyme and do not dissociate
- 4 types
- group-specific
- affinity labels
- suicide inhibitors
- transition-state analogs
- 4 types
True of False
The hydrolysis and removal of phosphates from ATP is a highly exergonic process
True
a lot of free energy is released
2 Forms
ADP + Pi = -30.5 kJ/mol (2 phosphates remain)
AMP + PPi = -45.6 kJ/mol (1 phospate remain)
Define Reversible Inhibitors
- molecules that bind non-covalently to the enzyme and can freely dissociate
- 3 types
- competitive, uncompetitive, and noncompetitive
- 3 types
Hydrolytic
Cleavage of bonds by the addition of water
What are Double-Displacement Reactions?
- one or more products must be released before all substrates can bind the enzyme
- results in a substituted enzyme intermediate, where the enzyme is temporarily modified
- substrates and products bounce on and off like ping pong balls
Draw a plot for Allosteric Enzymes
- allosteric enzymes do not show hyperbolic plots but show sigmoidal behavior
- require more complex kinetic models to interpret
Define Transition-State Analogs
- a type of irreversible inhibitor
- inhibitors that are designed to look like the transition state
- enzymes are designed to release maximal binding energy during binding of the transition state
- highly potent inhibitors b/c they maximize binding with the enzyme
Define Competitive Inhibition
- type of reversible inhibitor
- an inhibitor binds to the same site as the actual substrate
- an enzyme can bind to either a substrate to form ES or an inhibitor to form EI but it cannot bind to both (for an ESI complex)
- can increase KM but do not alter Vmax
What happens in Sequential reactions?
all substrates must bind to the enzyme before a product can be made
What are the two types of Sequential Reactions?
- ordered
- the substrates will bind to the enzyme in a specific order
- random
- the substrates can bind randomly ( in no particular order) to the enzyme
Group Transfer
Transfer of a functional group from one molecule to another
True or False
Thermodynamically unfavorable reactions are made favorable by the input of free energy from ATP hydrolysis
True
this is controlled by enzymes
What is a proton gradient?
- protons are pumped out of the mitochondial membrane (yellow cartoon) to generate an electrochemical gradient
- gradient is used by an ATP synthase (purple cartoon)
- ATP synthase pumps protons back in and uses the free energy of this reaction to generate ATP from ADP and Pi
What is the most efficient way that aerobic organism regenerate ATP?
Via the oxidation of fuels (photosynthesis in plants)
How do I get rid of Competitive Inhibition?
- can be relieved by increasing the concentration of substrate
- more substrate wil outcompete the inhibitor
- competitive inhibitors increase KM but do not alter Vmax
What is Oxidative Phosphorylation?
- one of the 2 pathways acetyl is shuttled through in Stage 3
- 4 pairs of electrons (8 total) generated in the citric acid cycle are used to make a proton gradient that shuttles electrons to O2 and generates ATP
What are the two types of Enzyme Inhibition?
Reversible Inhibitors
Irreversible Inhibitors
What kind of reaction is the following?
Lactate Dehydrogenase
- ordered sequential reaction
- electrons are transferred from NADH to reduce pyruvate to lactate
- NADH binds first, then pyruvate
- lactate is released first, then NAD+
What kind of buffer conditions change kinetic parameters?
- temperature
- enzymes become faster as the temp is increased but can unfold if temp is too high
- ionic strength
- adding more salt could weaken electrostatic interactions with a substrate
- pH
- enzymes can lose activity if certain amino acids are not properly protonated or deprotonated
Leonor Michaelis and Maud Menten developed a model that describes the behavior of the rate of enzymes. What is this model?
Where:
- E = enzyme (kept at a constant concentration)
- S = substrate
- ES = enzyme substrate complex
- P = product
- k1 = forward rate constant for formation of ES
- k-1 = reverse rate constant for decomposition of ES back to E + S
- k2 = forward rate constant for conversion of ES to P
Since we measure early near time zero, we do not worry about conversion of P back to S (k-2)
What kind of reaction is the following?
Creatine Kinase
- random sequential reaction
- transfers phosphate from ATP to creatine to form phosphocreatine and ADP
- substrates and products bind and release randomly
Isomerization
Rearrangement of atoms to form isomers
Your experiment shows your Vmax to be 44 micromolar per second. What is your turnover number given that you have 100nM enzyme in the assay?
440 s-1
Define Group-Specific Inhibitors
- a type of irreversible inhibitor
- they form covalent bonds with specific amino acids
- ex) DIPE reacts specifically with chemically reactibe serine residues
- helpful when trying to locate active site of amino acids
- ex) DIPE reacts specifically with chemically reactibe serine residues
What does a low KM and a high kcat describe?
An enzyme that binds substrate tightly and rapidly turns substrate to product is a highly efficient enzyme
What are some advantages of being able to inhibit enzyme activity?
- ensymes that are part of metabolic pathways are inhibited when the pathway needs to be shut down
- inhibitors are used as drugs to treat infections
- they target an essential enzyme from a pathogen
- inhibitors allow us to study enzyme mechanisms
True or False
Inhibitors can not alter KM or Vmax
False
Inhibitos can alter them individually or both
Activated carriers of two carbon fragments
- these molecules shuttle two carbon groups (acetyl) to where they will be used in metabolism
- most common is coenzyme A (CoA) which forms a thioester bond with the 2 carbon fragments
How fast can an enzyme work?
- the rate of an enzyme cannot be faster than the rate of diffusional control
- means that an enzyme rate is restricted by how often it encounters a substrate in solution
- limits enzyme to a maximum kcat/KM of 108 - 109 M-1s-1
- enzymes that can do this are said to have catalytic perfection
- every encounter with a substrate is productive and leads to product
What does Vmax reveal?
- reveals the turnover number of an enzyme
- how fast it converts substrate to product
- when Vmax is reached, this means that all enzyme is in ES complex
- Vmax = k2*Etotal
- k2 is also called kcat
- the turnover number and is what is reported in literature
- k2 is also called kcat
True or False
Fuel molecules such as glucose and lipids are complex, but the oxidation of these molecules occur one carbon at a time
True
These molecules get broken down into smaller fragments that are then oxidized to produce energy
What are the 5 Metabolic Principles?
- fuels are degraded and large molecules are synthesized, by a series of linked chemical reactions called metabolic pathways
- Adenosine triphosphate ATP is the metabolic energy source common to all life forms
- the oxidation of carbon fuels powers the formation of ATP
- although there are many metabolic pathways, they share a limited number of types of reactions and intermediates
- metabolism is highly regulated
NADP(H)
- nicotinamide adenine dinucleotide phosphate
- a major carrier of electrons that are donated to molecules during reductive biosynthesis
- differs from NADH in that is has a phosphate group attached to the 2’ carbon of the ribose sugar
- oxidized = NADP+
- reduced = NADPH
Define Catabolism
- a type of metabolism
- breakdown of fules to make useful cellular energy (ATP)
Fuel (carbohydrates, lipids) → CO2 + H2O + useful energy
True or False
Inhibitors can have different affinities for enzymes
True
some inhibitos are potent (strong binding) while some are weak inhibitors
Define Anabolism
- a type of metabolism
- use useful energy (ATP) to make complex molecules from simple precursors
useful energy + simple precursors → complex molecules
What are the units for velocity?
change in concentration over time
M/s
Oxidation - Reduction
electron transfer
What is First Order Reaction?
- when the reaction only depends on the concentration of A
A →P
V = k[A]
Define Suicide Inhibitors
- highly specific irreversible inhibitors
- inhibitor binds to active site as a substrate and is initially processed by the normal catalytic mechanism
- catalysis generates a reactive intermediate that then inactivates the enzyme
Define Enzyme Kinetics
- study of the rates of enzyme-catalyzed chemical reactions
- allows us to understant how efficiently an enzyme functions
Draw the Lineweaver-Burk Plot
What is the Michaelis-Menten Equation?
Write out the equation with corresponding graph
Where:
V0 = initial velocity V0 = (Vmax[S]) / (KM + [S])
Vmax = maximal velocity
[S] = substrate concentration
KM = Michaelis constant
- substrate concentration that results in 1/2 maximal velocity
What are Allosteric Enzymes?
- do not behave Michaelis-Menten kinetics
- can be positively or negatively regulated by molecules other than their substrates
- molecules will bind to places other than the active site to cause a conformational change
- results in cooperativity
- the binding of one substrate facilitates the binding of another substrate at a second active site
Activated carriers for reductive biosynthesis
- donate electrons to molecules to reduce them
What is Second Order Reaction?
- when two substrates are involved in a reaction
V = k[A]2
units for k is 1/Ms
A + B → P
