Term
| How are chemical rxns related to Enzms? |
|
Definition
| All chemical rxns in a cell are catalyzed by protein catalysts |
|
|
Term
| T/F Every enz is encoded by multiple genes |
|
Definition
| F - every enz is encoded by a unique gene |
|
|
Term
| What is standard state referring to? |
|
Definition
| Neutral ph, physiol temp and pressure |
|
|
Term
| What is a metabolic pathway? |
|
Definition
| Where the product of 1st enz is the substrate for the next enz in the pathway |
|
|
Term
| What form may a metabolic pathway take? |
|
Definition
| Linear, branched, cyclic etc... |
|
|
Term
| How were most major pathways elucidated structurally between 1935 and 1970? |
|
Definition
| Via Pulse Chase Radiolabelling |
|
|
Term
| What is pulse chase radiolabellign? |
|
Definition
| Give tissue or cell a free extract brief pulse of radiolabeled carbon 14, followed by a chase with non radioactive carbon 12. Need to give c-12 in order to be able to follow progress |
|
|
Term
| Other than PC-radiolabelling, how did pathways become elucdated in 1935 to 1970? |
|
Definition
| Via mutants - ability to identify what products weren't made if you knocked out specific areas. A-B-C-D, if mutant 1 is defective in enz 1, you know it can't convert A-B, and then do the same for other enzymes and find the sequence |
|
|
Term
|
Definition
| Attempts to assign function to every gene (and protein) w/ bioinformatics |
|
|
Term
| What is functional genomics? |
|
Definition
| Identifying the function of every gene in a sequenced genome |
|
|
Term
| What % of genes encode enzymes? what are the rest? |
|
Definition
| 90%, the rest are structural, mechical proteins. hormone, lectins, transport proteins, defense proteins etc... |
|
|
Term
| How many genomes have been sequenced entirely? |
|
Definition
|
|
Term
|
Definition
| Bioinformatic databse that attempts to reconstruct in silico an organisms metabolic pathways from its annotated genome sequences (contains 9m genes from 156 eukaryotes, 2000 bacteria and 133 archae |
|
|
Term
| How much would it cost to sequence the human genome today? |
|
Definition
|
|
Term
| What are modern forms of elucidation of pathway control? (list 5) |
|
Definition
| Assays through ENZ purification, Metabolomics, Subcellular fractionation, ultrafast tissue freezing and MCA |
|
|
Term
| What are 3 remarkable properties of enzymes as protein catalysts? |
|
Definition
Speed - Increase rate by 10^14 (lowers activation energy, no influence on delta G - increases rate at which EqM is attained)
Specificity - No side RXNS - Perfect lock and key
Control - Pacemaker enzymes control flux and movement |
|
|
Term
|
Definition
| the 3-D cleft on enzyme where S binds and conversion to P occurs. |
|
|
Term
| What is induced fit? ex. of hexokinase |
|
Definition
| Where the binding of a given S (glucose) leads to a conformation change in the enzyme that brings specific A.A. residues in active site into proper orientaiton with substrates |
|
|
Term
|
Definition
| Extra A.A. serve as a scaffold to create a 3-D active site from a few key A.A that are far apart in primary structure |
|
|
Term
| What is an important goal of protein structural biochemists? |
|
Definition
| Determining which A.A. residues participate in S binding and catalysis (is it polar, non polar, charged, aromatic) |
|
|
Term
| When could you possible encounter low specificity for an enzyme? |
|
Definition
| With digestive enzymes that degrade macromolecules (proteases and nucleases) |
|
|
Term
| What is the rule of thumb regardign pathway flux? why? |
|
Definition
| Keep flux low until the cell needs a given end product of the pathway. Many diseases result from loss of metabolic control |
|
|
Term
| What are non-protein enzymatic components that are required for catalytic activity ? |
|
Definition
Co-Factors
NOTE: NOT EQUAL TO ACTIVATORS |
|
|
Term
| What are the two major classes of co-factors? |
|
Definition
| Metal Cations and Co-enzymes (organic cofactors) |
|
|
Term
| What are examples of metal cations and where do they bind? |
|
Definition
Mg, MN, Zn
Bound at active site OR to substrate. |
|
|
Term
| how many enzymes require metal cations? |
|
Definition
| 1/3 of all enzymes, ALL KINASES |
|
|
Term
| What is the specific role of co-enzymes? |
|
Definition
| Transient carriers of specific atoms or functional groups |
|
|
Term
| When is a co-enzyme known as a prostehtic group? |
|
Definition
| If it covalently bound to an enzymes |
|
|
Term
| Can you have both a coenzyme and metal cation required? |
|
Definition
|
|
Term
Define:
holoenzyme
Apoenzyme
Ligan |
|
Definition
Holoenzyme - Enz + Cofactor
Apoenzyme - Enz - cofactor (inactive)
Ligand - any small molecule that can reversibly bind to a protein |
|
|
Term
| What two things determine how much ligand is bound to a given enzyme? |
|
Definition
1) Concentration of Ligand
2) Enz affinity for the ligand |
|
|
Term
| What is KD and what does low Kd mean? |
|
Definition
Kd = enz-ligand dissociation constant
Low kd means enz has a high affinity for the ligand |
|
|
Term
| How can you organize enzyme names? |
|
Definition
By trivial name (urease)
By Systematic name (describes type of rxn and substrate acted upon (urea aminohydrolase)
Enz commision (1 of 6 major classes) |
|
|
Term
| What are the 6 major classes of the enz commission? |
|
Definition
| Oxidoreductatses, transferaseae, hydrolases, lyases, isomerases, ligases |
|
|
Term
| What do each digit in the four digit code for ENZ commision represent? |
|
Definition
| Class, Subclass, Subclass-Subclass, Susbtrate type |
|
|
Term
|
Definition
| Enz variants that catalyze identical reactions, but are encoded by different genes |
|
|
Term
| T/f isozymes exist in different compartments or tissues |
|
Definition
|
|
Term
| Describe the isozymes of LDH |
|
Definition
| There are two different types, H and M chain. M type is muscle type and has 4 M chains, the kinetics favor the conversion of pyruvate to lactate during strenous exercise. The heart type LDH is four H- chain and it favor conversion of lactate back to pyruvate to clear lactate from blood during the recovery from burst muscle work |
|
|
Term
| What is the central role for isozymes |
|
Definition
| they provide eukaryotes with the machinery required for cell specific metabolism and metabolic control |
|
|
Term
| Why is it important to study the rxn rates of enz catalyzed reactions? |
|
Definition
| Provides us with information into enzyme mechanisms and help to define the role of an enzyme in vivo |
|
|
Term
| Pastuer's Vitalism theory? |
|
Definition
| Fermentation of sugar into alcohol is catalyzed by ferments, which are inseperable from the structure of the living yeat cell |
|
|
Term
| What did buchner discover wrt fermentation? |
|
Definition
| That it was due to molecules that continue to function when removed from cells |
|
|
Term
| Who established the modern era of ENZ kinetics? |
|
Definition
|
|
Term
| How did Maud menten create protein separation? |
|
Definition
| The use of electrophoresis to separate proteins by size and charge |
|
|
Term
| Why were michaelis and menten techniques so revolutionary? (2) |
|
Definition
1) They used buffers to control rxn ph
2) Measures the intitial rate at various subtrate concentrations and constant enzyme concentration. This lead to greatly simplified results, no substrate depletion or end product inhibition |
|
|
Term
| How did M and M propose rxn mechanisms differently than before? |
|
Definition
| Proposed the existence of both fast and slower (rate limiting) steps. like how sucrose binding to enzyme is fast, but then sucrose separation is slow |
|
|
Term
| List the four types of Enz assays? |
|
Definition
| Radiometric, Fluorescence based, HPLC based, Spectrophotmetric |
|
|
Term
| What are spectrophotometric assays? |
|
Definition
| If the S or P absorbs light at a specific wavelength, you can use the spectrophotometer to measure change in ABS/min and then convert this into (micro mol S into P/min) using Beer's Law (requires S or P's molar extinction coefficient) |
|
|
Term
| What is the MM equation? What does it relate |
|
Definition
Relates Substrate concentration to the Velocity of an enz catalyzed rxn
Vo= (Vmax x. [S]) / (Km + [S]) |
|
|
Term
|
Definition
| The max catalytic rate, i.e. when enzyme is saturated with Substrate |
|
|
Term
| what are the units of vmax? |
|
Definition
| Units are usually mmol p produced/min/mg of enz protein |
|
|
Term
|
Definition
Defined as the turnover #
The number of S molecules converted to P/sec, per molecule of Enz when it is saturated with S. |
|
|
Term
| What are the units for kcat |
|
Definition
|
|
Term
|
Definition
|
|
Term
| Give an example of an enzyme with a ridiculous kcat |
|
Definition
|
|
Term
| What is the Km? what does it show? |
|
Definition
The MM coefficient
Shows the substrate concentration that will yield the 0.5vmax for a given enzyme |
|
|
Term
|
Definition
|
|
Term
| Is km a constant for all enzymes? |
|
Definition
| No, a given enzyme has a constant Km |
|
|
Term
| What are the fixed constant for any enz that we try to figure out? |
|
Definition
|
|
Term
| what is catalytic efficiency and how do you calculate it? |
|
Definition
| How good of a catalyst the enzyme is, calculated by (Kcat/Km) or (Vmax/Km) |
|
|
Term
| What are two ways to determien Vmax and Km? |
|
Definition
1) Estimate from plot of Vo and [S]
2) Use ENZ kinetics software to directly fit Vo vs. [s] data to M-M equation (far better option) |
|
|
Term
| What enzyme could exhibit non MM kinetics? |
|
Definition
| Only multimeric (quaternary) structured enzymes |
|
|
Term
| What is true about function and structure in complexity? |
|
Definition
| The more complex function of a protein requires a more complex and larger native structure |
|
|
Term
| What is positive cooperativity? |
|
Definition
| A form of non MM kinetics, where binding of substrate leads to conformational change in enzyme and a sigmoidal plot (not hyperbolic) |
|
|
Term
| What happens when one substrate binds to a enzyme with sigmoidal kinetics? |
|
Definition
| The Enz converts from Tense state (where it has low affinity) to a relaxed state (where it has high affinity for S) as it is stabilized by addition of S |
|
|
Term
| What two things can reversibly bind to promote a change in enzyme conformation for cooperative enzymes? |
|
Definition
| Inhibitors and Activators |
|
|
Term
| What are inhibitors and activators otherwise known as? |
|
Definition
|
|
Term
| Where do Act and Inh reversibly bind? |
|
Definition
| To 3-D ssites - Allosteric Sites |
|
|
Term
| What are enzymes that can be modiied by Act or Inh called? |
|
Definition
| Allosteric enzymes, or cooperative enzymes |
|
|
Term
| Describe what happesn to Aspartate Transcarbamylase after interaction with effector? |
|
Definition
| It changes from bunched formation to open ball formation, to allow greater affinity |
|
|
Term
|
Definition
| The allosteric modulator is the substrate alone, and this results in sigmoidal S saturation |
|
|
Term
|
Definition
| Allosteric modulator is not the substrate, and can result in either activation or inhibition |
|
|
Term
| Are both homotroic and heterotropic enzymes considered allosteric? |
|
Definition
|
|
Term
| Do pacemakers enzymes interconvert between MM and sigmoidal kinetics? |
|
Definition
| Yes, example of pyruvate kinase from cyanobacterium |
|
|
Term
| Describes how pyruvate kinase interconverts its enzyme kinetics? |
|
Definition
W/o allosteric effectors, it shows MM kinetics. But with, it shows sigmoidal. Example of G6P, that causes it to become MM from sigmoidal. (G6P in this case is an activator, as the km is lowered).
ATP is an inhibitor as it increases Km |
|
|
Term
| How do you determine the kinetic constant with cooperative enzymes? |
|
Definition
|
|
Term
|
Definition
If =1, there is no cooperativity, therefore S0.5 =km
If > 1.0 there is +ve cooperative, leads to sigmoidicity of Vo vs. [S} plot. An inhibitor would cause NH to be much greater than 1, as it significantly decreases Km |
|
|
Term
| What is the significance of Sigmoidal Kinetics? |
|
Definition
| Allows metabolite effector to cause a large shift in Vo. With activator, typically get MM kinetics and decrease Km, and with inhibitor, nH goes up, Km increases. |
|
|
Term
| What are effectors of ENZ activity used for? |
|
Definition
| For fine tuning of allosteric ENZ activity - not total on/off control |
|
|
Term
| What are the two types of ENZ inhibition? |
|
Definition
| Competitive and mixed competitive inhibition |
|
|
Term
| What is feedback inhibition and what is it's role? |
|
Definition
| When the product of a pathway feedback and inhibits enzyme that catalyzes the committment step of a pathway. Role to help balance pathway flux w/ cell's need for pathway's end product (s). Important in the fine control of metabolic pathways in vivo |
|
|
Term
| Describe Comp inhibition (km and Vmax) |
|
Definition
I and S compete for active site, where I looks like S.
Comp. I increases the Km for a S, but it can be reversed by high levels of [S] and thus doesn't affect vmax (overcome by saturation) |
|
|
Term
|
Definition
| The enzyme inhibitor dissociation constant, a low ki value means that its an effective inhibitor. |
|
|
Term
|
Definition
|
|
Term
| What is mixed comp inhibition? |
|
Definition
I binds to free E or ES complex at the allosteric I site
I binding causes a change in conformation which interferes with enzyme and substrate interactions |
|
|
Term
| To which enzyme types does mix comp inhibiition particularly apply? |
|
Definition
| Allosteric pacemaker enzymes |
|
|
Term
| Describe effect of Km and Vmax due to mixed comp inhibition |
|
Definition
Decreases Vmax, and increases it Km (interferes with Sbinding to Enz)
Can't overcome inhibition by increasing substrate |
|
|
Term
| What determines how effective the inhibitor is? |
|
Definition
| The Ki value - a low K value = potent inhibitor |
|
|
Term
|
Definition
Use a shortcut, determine the I 0.5 value
The inhibitor concentration that yield half maximal inhibition. If the i0.5 is low, it is a potent inhibitor.
Done by fixing the [S] to approximately the Km value |
|
|
Term
| How do activators affect Enz interactions? |
|
Definition
| Increase Vo by binding to an allosteric activator site that causes a change in conformation that promotes ENZ and S interactions |
|
|
Term
| Describe the catalytic and regulatory subunit model? |
|
Definition
| Some enzymes have both C and R subunits. The modulator (activator) binds to the R subunit, causing a conformational change in the C subunit, allowing it to better bind the substrate (Tense to Relaxed form) |
|
|
Term
| How does A affect Km? Vmax? I binding? |
|
Definition
It typically decreases Km or S0.5.
May (?) increase Vmax, and interfere with I binding |
|
|
Term
| What three things do you always need to know about a given A? |
|
Definition
1) Effect on Vmas and Km
2) A0.5 Value (done by putting S to km level)
3) Influence on Enz-I interactions |
|
|
Term
| Describe the action of ASP and GLU to PYR kinase? |
|
Definition
| GLU is a mix comp inhibitor and thus increase km, but ASP is an allosteric activator. However, ASP also reverses GLU inhibition and when both are present, the levels of activity are higher than baseline levels |
|
|
Term
| Why is T so important to ENZ? |
|
Definition
| It has a huge impact on species distribution and diversity, and almost every asepct of their physiology and biochem. Also helps us to predict how future temp increases due to global warming may impact the distribution and physiological status of specific animal and plant species |
|
|
Term
|
Definition
| An specific range of T where a species prefers to live. Antartic fishes can live from -2 to +2, but die of heat shock at greater than 4. Marine molluscs can tolerate changes of >30 degrees/day |
|
|
Term
| What's the most thermally tolerant eukaryote/ |
|
Definition
| Alvinella pompejana - survive with one end of its body 60 degree hotter than its other end |
|
|
Term
| What is the relationship between T and vmax? |
|
Definition
| Usually increase Vmas with increase in T, when enz is stable |
|
|
Term
|
Definition
| The Temp coefficent of the reaction |
|
|
Term
|
Definition
| Vo (T + 10degrees) Vo (T) |
|
|
Term
|
Definition
| Around 2.0, meaning that with increases of 10 degrees will double reaction rates |
|
|
Term
| Does T alter both Vmax and Km? |
|
Definition
|
|
Term
| Describe Three key aspects of ENZ adaption to T |
|
Definition
Thermal Stability of Extremozymes
Q10 values
Thermal Kinetic windows |
|
|
Term
| What are the species that can inhabit boiling hot springs? |
|
Definition
|
|
Term
| What are the types of Extremophiles? |
|
Definition
| Thermophiles, halophiles, acidophiles, basophiles |
|
|
Term
| What are the main issues with living in the abyss, greater than 3km of depth? (4) |
|
Definition
Very high Pressure,
Constant Darkness,
Hypoxia, Food and cold |
|
|
Term
| Where do you typically find hydrothermal vent ecosystems? First discovered |
|
Definition
| On spreading tectonic plates on ocean floor, first discovered in 1979 |
|
|
Term
| What are chemoautotrophs and where are they found? |
|
Definition
| Microbes at base of food chain in hydrothermal vents, oxidize reduced metals (like H2S) as an energy source |
|
|
Term
| Can animals lack Digestive systems? |
|
Definition
| Yes, if they have symbiotic chemotrophic microbes |
|
|
Term
| T/F the biomass per m2 of a hydrothermal vent system is much larger than the tropical rainforest or coral reef |
|
Definition
|
|
Term
| Issues with studying hydrothermal vent specie? |
|
Definition
1) sample collection
2) Lab conditions (120 c and 1000 atm)
3) Weird morphology |
|
|
Term
| Describe one example of extremozymes to biotech? |
|
Definition
| Taq DNA polymerase to allow cooling and annealing of DNA strands at high T levels |
|
|
Term
| What are three reasons for increased thermal stability of extremozymes? |
|
Definition
1) Increased # of charged A.A's, increases # of ionic bonds
2) Increased # of hydrophic A.As - strength of hydrophic interactions increases
3) Less uncharged polar A.A - decreases number of H bonds, |
|
|
Term
| How does h bond strength relate to T |
|
Definition
| It decreases with temp Increases |
|
|
Term
| What do low Q10 values tell us? |
|
Definition
| Enzymes have adapted well to low temps (poikilotherms) |
|
|
Term
| What are thermal kinetic windows (TKWs) |
|
Definition
| Temp range over which ENz's max affinity for a S occurs. Physiological temp range for that organism |
|
|
Term
| Describe the TKWs for LDH of different animals? (same for Hydroxypyruvate reductase) |
|
Definition
| Each animals has different thermal niches, and thus the lowest Km for the animals LDH occur in the physiological temperature range for each species. Same enzyme, different species and different Km constants |
|
|
Term
| What is the effect of pH on ENz? |
|
Definition
They have characteristic pH activity optima
Usually from 5-8.
pH optima in vivo is related to location (pepsin is optima at 1.5) |
|
|
Term
| Why does vo of Enz change with pH change (3)? |
|
Definition
1) Degree of ionization of charged A.A side chains (lose ability to associate with substrates)
2) Degree of ionization of Substrate molecules
3) Irreversible denaturation of enzymes |
|
|
Term
| Why do ENZ purification? (4) |
|
Definition
| 1) Eliminate metabolites that could interefere with kinetics and structual studies. 2) Allows accurate determination of ENZ physical properties 3) Allows antibody production against purified ENZ 4) Partial A.A. sequencing of purified ENZ subunits |
|
|
Term
|
Definition
| Extract tissue in buffer solution and then centrifuge to obtain crude extract (1,000 of different proteins and metabolites) |
|
|
Term
| What is the goal of purification? |
|
Definition
| to isolate a single ENZ from a crude extract |
|
|
Term
| What is considered a lot of purified enzymes? |
|
Definition
|
|
Term
| List the steps of creating an extract? |
|
Definition
| Extract tissue in buffer, use polytron homogonzier, use FPLC, purify based on electrophoresis |
|
|
Term
| If an organism has hundreds of similar enzymes, how do know when it's pure? |
|
Definition
| A single line on the electrophoresis |
|
|
Term
| T/F the cell [ENZ] <<< In vitro assay |
|
Definition
| F! The cell houses enzymes at a very high concentration |
|
|
Term
| What is the dilution problem? |
|
Definition
| The protein concentration is very low, and dilution tends to cause subunits of oligomeric enzymes to disassociate |
|
|
Term
| What are two possible solutions to the dilution problem? |
|
Definition
Do large scale ENZ prep so [protein] remains high
- Add protein aggregating reagent to buffers (glycerol) to mimic in vivo conditions, so that oligomers aren't dissociated |
|
|
Term
| Why do we use all approaches and preparation to study metabolism? |
|
Definition
| We because it is very complex and we must compromise. we must show that in vitro enzymes actually work in vivo to control metabolism |
|
|
Term
| What is thermodynamics? Bioenergetics? |
|
Definition
| T- Quantitative study of energy transformation in matter Bio - same, but in living systems |
|
|
Term
| What does Gibbs energy tell us? |
|
Definition
| Prediction of direction and equilibrium position of a reaction |
|
|
Term
|
Definition
| The energy capable of doing work as it proceeds to equilibrium (at ph =7) |
|
|
Term
| System not at equilbrium have G'? |
|
Definition
| Those system far from EM proceed in direction of negative delta G |
|
|
Term
|
Definition
| 0, no further free energy change can occur |
|
|
Term
| How are G and G' related? |
|
Definition
| By G = G' + RTln [B]/[A]. |
|
|
Term
| What is the mass action raio? |
|
Definition
| The B/A in free energy equation |
|
|
Term
| What does it mean when mass action ratio =1 |
|
Definition
That B/A =1
If Keq = 1, then system is at equilibrium |
|
|
Term
| What does it means when mass action ratio >1.0 |
|
Definition
| if Keq =1, then the reaction will be exergonic and spontaneous |
|
|
Term
| What does it means when mass action ratio <1.0 |
|
Definition
| If Keq = 1, then the reaction and nonspontaneous in the given direction and endegonic |
|
|
Term
| T/f do time or rate catalysts affect G? |
|
Definition
|
|
Term
| Does time of reaction relate to how negative it's delta G value is? |
|
Definition
| No, a reaction like ATP hydrolysis has highly -ve G, but it won't occur until it's Ea is overcome. Still is spontatenous, but that doesn't equal fast |
|
|
Term
| what are mass action ratios for typical pacemaker ENZ? |
|
Definition
| They often catalyze non equilibrium reaction in vivo (where the Keq is much greater than the Mass actio ratio) and the G is much less than 0 |
|
|
Term
| Describe the additive nature of free energy change? |
|
Definition
To make an endergonic rxn possible, it is often coupled to an exergonic rxn
like glu - G6P (endergonic) and then ATP to ADP (exergonic): net is exergonic and favourable |
|
|
Term
| If a rxn is close to EM is it irreversible? |
|
Definition
| No, it is freely reversible. IT is those that are far from EM that irreversible (think about one directional committed step enymes) |
|
|
Term
| What are the enzymes that control pathway flux? |
|
Definition
| Pacemaker, rate determining step enzymes |
|
|
Term
| What is classical approach to studying metabolic control (3 steps) |
|
Definition
| Idenitifying, purifying ,and characterizing the key non EM or pacemaker enzymes, as they generally control overall pathway flux |
|
|
Term
| What are two methods to identify the pacemaker enzymes of a given pathway? |
|
Definition
A) Circumstantial evidence - the placement of an enzyme in a pathway
B) Direct evidence, done by measuring Delta G of a reaction |
|
|
Term
How does PFK's KEq relate to it's mass action ratio?
what does this tell us |
|
Definition
Much larger KEq, 1500x larger - threfore G is much less than 0 and it is greatly displaced from EM
Tells us its a likely pacemaker enzyme |
|
|
Term
| How efficient is glycolysis? |
|
Definition
| Only 38% efficient, lose 60% as heat dissipated to environment, which gives it a thermodynamic push (you actually need it to be inefficient) |
|
|
Term
| What is metabolic control analysis? |
|
Definition
Biological system can display properties that are not possessed by isolated components (in vitro not = to in vivo)
Tries to quantify relative contribution of each ENZ to overall control of pathway flux in the intact system |
|
|
Term
| What does MCA predict, as opposed to traditional pacemaker purification studies? |
|
Definition
| Control of flux is shared by many, if not all, enzymes in a pathway. and That the degree of control exerted by each enzyme changes as physiological status of the organism changes (fed vs. starved, exercise vs. rest) |
|
|
Term
| What is the flux control coefficient? |
|
Definition
| Describes the amount of flux change (J) relative to small changes in activity of a specific enzyme |
|
|
Term
| What is the range of the CJe? |
|
Definition
|
|
Term
| What does CJe close to 1.0 say?Close to 0 |
|
Definition
The change is flux is almost proportional to change in [E] and thus it is very important.
Close to 0 is opposite |
|
|
Term
|
Definition
Change enzyme [] by mutagenesis or genetic engineering
Determine flux in vivo (difficult task) |
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Term
| Why is it so important to understand the structure and control of metabolic pathways (2)? |
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Definition
Understanding health disease due to metabolic disorders
Ability to do effective biotech via metabolic engineering |
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Term
| What are the energy reservoir of most cells? |
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Definition
| Large insoluble organic fuels (polysac and lipids) |
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Term
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Definition
| Glycogen starch, triglycerides |
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Term
| Where does energy come from in polyglucans |
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Definition
| Contain many high energy covalent bonds, and the energy in those bonds are released to fuel other things |
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Term
| How is energy generated from fuels? |
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Definition
| Through oxidation to smaller molecules via sequential enzyme catalyzed reactions |
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Term
| Where do we harness the energy from biochemical reactions? |
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Definition
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Term
| Describe ATP and why it is a energy reservoir? |
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Definition
Has 2 high energy phosphoanhydride bonds, that are neither weak nor unstabe. You need a mg cofactor always.
The reason it is so favorable is because hydrolyzing the bond removes the negative forces of repulsion between the phosphate groups |
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Term
What is the actual in vivo G of ATP?
Proposed G? |
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Definition
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Term
| List 6 roles of ATP as a fuel |
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Definition
1) biosynthesis
2) Mechanical work
3) ion pumping 4) signal transduction
5) Bioluminescne
6) thermogenesis |
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Term
| Describe ATPs role in luciferase? |
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Definition
| With ATP, in vitro, the tobacco plant is able to glow |
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Term
| Why do we need biolumiscnece? |
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Definition
Due to the extreme conditions in the ocean's abyss
Used for luring prey, finding mates, camoflouge and decoys |
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Term
| What are the two ways a cell can make ATP? |
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Definition
| Through subtrate level phosphorylation and chemiosmotic coupling |
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Term
| What is substrate level phosphorylation |
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Definition
| Transfer of P from ADP to ATP via kinases |
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Term
| What is chemiosmotic coupling? how does it work |
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Definition
Respriatory electron transport chain.
They perform redox reactions (as they transfer high energy electrons to the more and more electronegative atoms).
This generates high H+ concentration and a proton motive force, which drives back through an ATP synthase to create ATP |
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Term
How is ATPs bond Energy transferred into biosynthetic routes?
What enzyme does this? |
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Definition
Via other Nucleoside triphosphates (NTPs)
eg. dATP dGTP dTTP dCTP.
The enzyme that accomplishes this is Nucleoside diphosphate kinase |
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Term
| What does nucleoside diphosphate kinase do? |
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Definition
Transfer bond energy in ATP to other routes via NTPs
ATP + NDP <-> ADP and NTP |
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Term
| How do pacemaker enzymes control ATP levels? |
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Definition
| If ATP levels significantly drop due to utliization, the production rate will increase, as the steady state ATP [] should be a constant |
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Term
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Definition
| NO! IT is an energy transmitter |
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Term
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Definition
| Very fast - Each E.coli has 5,000,000 ATP molecules, but that only does 2 sec of work. A human process 40kg of ATP/day |
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Term
| What happens in the final stage of macromolecule synthesis with regards to NTPs? |
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Definition
NTPs are hydrolyzed by PPi cleavage
dNMP + dNTP --> d(NMP)n+1 + PPi |
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Term
| What does DNA polymerase do at the end of macromolecule synthesis? |
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Definition
| It adds a nucleotide base to DNA polymer, thus create inorganic pyrophosphate |
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Term
| What about the dogma and PPi is categorically wrong? |
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Definition
| That all PPi is hydrolyzed by inorganic PPIase |
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Term
| What actually happens with PPi in animal cells? |
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Definition
| In animal cells it doesn't build up, it is constantly hydrolyzed to inorganic phosphate (Pi) |
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Term
| What happens to PPi in plant cells? |
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Definition
| there isn't an excess of PPIase, so PPi is allowed to build up, and then subsequently used in various cellular processes as alterantive to ATP during a starved energy state |
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Term
| T/F there are alternative enzymatic pathways to use PPI instead of ATP? |
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Definition
| Yes! Instead of using ATP PFK, a plant cell can PPi PFK during a stress |
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Term
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Definition
It is a key biochemical adaption of many microbial and all plant species to abiotic stress that deplete cellular ATP like Anoxia.
I.E. PPi conserves ATP! |
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Term
| What is the alternative to H+ATPASE in cell vacuole for PPi? |
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Definition
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Term
| Which of ATP and PPI likely evolved first? |
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Definition
| PPi was actually likely life's first energy transmitter |
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Term
| Why is ATP preferred to PPi? (2 reasons) |
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Definition
1) ATP hydrolysis releases more energy as there are two high energy phosphoanhydride bonds
2) ATP and other NTPS are more chemically versatile |
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Term
| Why is ATP (and other NTPS) more chemically versatile than PPI? |
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Definition
| Beause it can directly contribute to DNA or RNa, and NADPH synthesis. (has adenosine backbone) |
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Term
| What are the three roles of catabolism? |
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Definition
| To create ATP, Reducing power, and to yield low molecular weight building block materials |
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Term
| What is the role of the nicotinamides? |
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Definition
| They function with dehydrogenases as soluble electron carriers |
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Term
| Is NADPH oxidation endergonic or exergonic? |
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Definition
| Exergonic, that's why it is favourable |
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Term
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Definition
| Enzymes that use either NADP+ or NAD (P) H |
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Term
| What is an amphibolic pathway? |
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Definition
| A pathway that can function in catabolic or anabolic direction |
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Term
| What is the main issue with amphibolic pathways? |
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Definition
| Futile Cycles, and having to control forward direction without controlling backwards direction |
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Term
| What is the role of pacemakers in avoiding futile cycle? |
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Definition
| They are maintained at non EM levels that only goes one direction |
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Term
| How do you solve the issue of futile cycles - most important method |
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Definition
| Have the same product act as an activator to one direciton and an inhibitor to the other (Temperol Separation) |
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Term
| What is the secondary, less useful way to solve futile cycles/ |
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Definition
| Physical separation, where the catabolic and anabolic pathways separated by organellar compartmentation |
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Term
| Give an example of organelle compartmentation |
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Definition
| Fatty acid degration occurs in mitochondria, but biosynthesis occurs in the cytosol |
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Term
| What are cyclic pathways? |
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Definition
| Small circular pathways that are able to link catabolic and anabolic pathways |
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Term
| What is the role of anapleurotic pathways? |
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Definition
| To fill up or replinish cyclic pathway intermediates that have been drained for biosynthesis |
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Term
| What is anaplerotic carboxylase? |
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Definition
| Enzyme that helps to maintain high [] of TCA cycle intermediates |
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Term
| What is the general rule about flux and a metabolic pathway? |
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Definition
| Flux of metabolites through any pathway must be controlled so that its' only as great as required |
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Term
| What 2 major types of metabolic control exist? |
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Definition
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Term
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Definition
| Slow, expensive, and mostly done by synthesis or degredation of whole molecules |
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Term
| When is coarse control very important? why? |
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Definition
| During long term adaptive or developmental changes,because it replaces abnormal or damaged enzymes |
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Term
| What is the general correlation between mRna [] and change in [] of proteins? |
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Definition
| Only about a 50% correlation between changing the conc of mrna and a subsequent change in the concentration of protein |
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Term
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Definition
| The ability to recognize why there is a lack of correlation between in vivo levels of mRNA and the protein it encodes |
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Term
| What are two main reasons why there is a lack of correlation between mrna and protein levels? |
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Definition
MicroRNA - small pieces of mRNA that bind to mRNA and lead to repression of translation
Riboswitches - pieces on the primary transcript of mRNA that bind to metabolites and causes an expression of genes that help to degrade the metabolite (i.e. fat) that its bound to |
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Term
| What is ubiquitin known as? |
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Definition
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Term
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Definition
| It tages specific lysine residues |
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Term
| What occurs if something is polyubiquinated? |
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Definition
| It is taken through the 26s proteasome death star and destroyed |
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Term
| describe 5 key aspects to the PolyUB toolkit? |
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Definition
| E1 activate, E2 conjugase, E3 ligase, UB, and the proteasome |
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Term
| What is the correlation between UB and human disease? |
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Definition
| Many neurodegenerative disorders arise from accumulation of protein granules, i.e. defects in UB proteasomal machinery |
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Term
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Definition
| Energetically cheap, controls momentary needs of the cell, and mostly applicable to pacemaker enzymes |
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Term
| What is the least important (in relations to the others) Fine control mechanism? |
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Definition
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Term
| Describe how [S] can affect pathway control? |
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Definition
| A increase in 2-3x of [S] can leads to change in 100x for pathway flux |
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Term
| IS the substrate [] in vivo usually saturating? |
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Definition
| No, typically subsaturing, at km |
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Term
| Give an example of pH fine control? |
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Definition
| Dark to light transition in algae occur when ph is shifter from 7 to 8. This leads to promotion of light activity and calvin cycle enzymes |
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Term
| T/F all pacemaker ENZ are controlled by multiple effectors? |
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Definition
| T - effectors can dramatically change pathway levels |
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Term
| What is most important about activators and inhibitors in a pathway? |
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Definition
| The ratio of A:I, as this will determine how much V changes |
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Term
| What are adenylates and how do they factor in fine control? |
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Definition
| They are AMP and ATP and they control rate of ATP formation. This is potentially a huge change, as a small change in ATP can lead to a enormous change in AMP. high sensitivity to ATP changes |
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Term
| Describe the example of locust flies and ATP and AMP |
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Definition
| AMP activates PFK to create more ATP, while it inhibits the FBPase (backwards glycolysis) ATP inhibits PFK because if you have ATP, you don't need PFK to make more |
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Term
| What is the general model of PTM's? |
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Definition
| To convert a less active ENZ with more active form |
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Term
| Is major mechanism of PTM controlled by internal or external stimuli? |
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Definition
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Term
| What is the major mechanism of reversible covalent modification? |
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Definition
| Phosphorylation - dephosphorylation |
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Term
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Definition
| It can either activate or inhibits complete target ENZ activity and controls nearly every aspect of cell biology |
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Term
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Definition
| Krebs and Ficher, when examining glycogen phosphorylase |
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Term
| Describe P/deP in glycolysis? |
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Definition
| ENz are turned when dephosphorylated, and on when they become phosphorylated |
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Term
| What kinds of intracellular signals exist? |
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Definition
| AMP - becomes an intracellular signal that activates AMP K, favors catabolism |
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Term
| What are major form of extracellular signals? |
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Definition
| Anything that leads to cellular second messengers |
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Term
| What is the role of AMPK? |
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Definition
With high AMP, it inhibits anabolism (form ADP from aTP) and activates catabolism (Form ATP)
. Note that adenyalte kinase takes ADP and turns it into AMP |
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Term
| How does P/deP prevent futile cycle? |
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Definition
| The same signal shuts off on half of the cycle and activates the other |
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Term
| What is the opposite of a protein kinase? |
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Definition
| PhosphoProtein phosphotase |
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Term
| Are the Kinase and phosphotases subject to their own controls as well as controlling other things? |
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Definition
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Term
| What causes cancer, diabetes, arthritis and cholera? |
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Definition
| Abnormal protein phosphorylation |
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Term
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Definition
| A pair of human protein kinases that control cell proliferation, survival and differentiation. With high ERK, this lead to altered protein phosphorylation in brain neurons and link to cancer (after being exposed to cell phone waves) |
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Term
| What is the cellular important of Disulfide Dithiol interconversion? |
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Definition
| IT links the photosynthetic E.T.C to the light activation of calvin cycle enzymes. With disulfide, in an oxidizign environment, they are inhibited (dark), but light leads to reduction and dithiols and activations |
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Term
| What is the role of monoubiquination? |
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Definition
| Reversible PTM that is non destructive and reversible, unlike poly UB. Influences protein interactions and locations. LEads to control of endocytosis, transcription, and one example of metabolic pacemaker enzyme. |
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Term
| Describe the given example of monoubiquination to a pacemaker enzyme? |
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Definition
| PEP carboxylase. Is an anaplerotic pacemaker enzyme that exists in all cells. When monoubiquinated is becomes inhibitory as the PEPC's km increases. However, when non ubiquinated, it is active. |
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Term
| What is PEPcase related to? what is the takehome message? |
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Definition
| Castor Beans and Ricin, that the same pacemaker enzymes can show tissue specific PTMs. It can be either phosphorylated and activated, or monoubiquinated and inhibited. |
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Term
| Describe Subunit Assocation Dissociation |
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Definition
| Small effector molecules binds to specific subunits and causes aggregation or dissociation to yield active enzyme. Multimeric enzymes are less active when dissociated. |
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Term
| Give an example of subunit association dissociation? |
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Definition
| The active form of Acetyl-COA is favored in the fed state, but in the starved form, the various subunit monomers dissociate. |
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Term
What occurs with the pyruvate kinase from pituitary glands?
What is moonlighting |
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Definition
| When active, it is a homotetramer, but when reduced an inactive, it gains thyroid hormone binding activity (moonlighting, is a multifunctional enzyme) |
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Term
| What is the role of reversible association of metabolons? |
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Definition
| That in low flux, starved environment, the physical distance between enzymes leads to lower rate of formation of product (Vo). However, in high flux the various enzymes associate together into a metabolon (5' structure) |
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Term
| What is an ambiquitous enzyme? what does it bind to? |
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Definition
| An enzyme that is part of a metabolon and binds to particulate sstructures (membranes, cytoskeleton), depending upon cell's metabolic/physiological status |
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Term
| What controls rev. associations of metabolically sequential ENZs? |
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Definition
| Reversible covalent modifications , substrate [], change of pH, effectors |
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Term
| What are the three advantages of metabolons? |
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Definition
1) Decreases diffusion time of P which serves as S for next ENZ (Channelled)
2) Enz: ENZ interactions may alter kinetic constants
3) No need for large (free intermediates) - cell limited solvent capacity - not taxed |
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Term
| Whats a good example of quinary structure? How does it work? |
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Definition
Krebs TCA cycle enzymes
- In on form (light) the calvin cycle enzymes associate, and in off, they dissociate for low flux |
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Term
| Give an example of certain cytoskeletal proteins where metabolons associate? Why is this important? |
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Definition
| During anaerobic muscle work, a glycolytic metabolon forms on F-actin and myoin, in order to create massive amounts of ATP for power contractions. It increases the respiration rate accompanied by the formation of metabolon, that channesl pyruvate into mitochondrai to revert to aerobic respiration |
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Term
| Describe PEP carboxylase and Metabolons |
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Definition
| The PEP carboxylase forms anaplerotic metablons with other glycolytic enzymes on the surface of mitochondria |
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Term
| What are the three faces of Enzyme? |
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Definition
| Catalytic, regulatory and social |
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Term
| What are the four main biochemical transducers? (link external stimuli with a coordinate intracellular response) |
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Definition
| cAdenylates (cAMP and cGMP), Ca2+, DAG and IPS, and oligosacchardies (only in plants) |
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Term
| What makes cAMP from ATP? |
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Definition
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Term
| What converts cAMP to AMP? |
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Definition
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Term
| Describe cAMPs activation for PKA? |
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Definition
| Camp binds to regulatory surface, and it changes PKA catalytic surface, making it active |
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Term
| Does AMP affect PKA? What does AMP do? |
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Definition
| No. It activates AMPK, which favors ATP formation when ATP is low |
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Term
| What does adenyl cyclase do? |
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Definition
| Catalyzes the formation of caMP after an external stimuli - it is activated directly through GcPR subunit dissociation |
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Term
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Definition
| Ca binds to Calmodulin (CaM) and it fulfills the role of a second messenger. Stimulus leads to a spike in calcium, and calcium binds to Calcium binding proteins which then bind Enzs and trigger physiological responses |
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Term
| Do plants or animals have more Ca2+ binding proteins? |
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Definition
| Plants! they have >50, we only have one. |
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Term
| How exactly are the effects of Ca second messengers carried out? |
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Definition
| They mediate effects through positive activations of protein kinases and negative inhibition of phosphoprotein phosphotases |
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Term
| What % of eukaryotic genes encode protein kinases? |
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Definition
| 2-6% of genes, >1000 different kinase isozymes in a eukaryotic cell |
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Term
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Definition
| The estimated # of different protein kinase genes |
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Term
| List, if you wanna have a good time, the 6 steps to stimulus response coupling? |
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Definition
Generation of signal
Signal transmission (external)
Signal Reception
Signal Transduction (intracellular)
Amplification
Signal Terminaiton by decd [2nd messengers] |
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Term
| What is the Ca Singature hypothesis? |
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Definition
| that the spatial - or temporal - magnitude of intracellular calcium concentrations change would dictate a specific cellular response (not really confirmed) |
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Term
| What is an amphipathic molecule? What signalling molecules fit this descriptions |
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Definition
One with both polar and non polar components
Phopholipids that break down into DAG and IP3 |
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Term
| Describe the role of Phospholipase C in cellular signalling? |
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Definition
| Breaks down Phosphatidylinisotol (glycerol + 2 fatty acids + phosphate alcohol) into DAG (Diacylglycerol) and IP3 (Just phosphate alcohol) |
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Term
| Describe the signalling cascade produced by formation of IP3 and DAG? |
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Definition
| DAG directly binds to an activates Protein Kinase C, while IPs binds to a channel and leads to a release of Calcium, which binds to and activates PKC |
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Term
| What two things directly activate PK-C? |
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Definition
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Term
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Definition
| Internal signal transduction mechanism. it phosphorylates cellular proteins and creates cellular response to the inital external stimuli |
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Term
| What is the correlation of disease to Pk-c? |
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Definition
| Many toxins and disease lead to incorrect signal transduction and incorrect protein phosphorylation. Incorrect phosphorylation by Pk-C. Increase [cAMP] and keep Pk-A always on |
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Term
| Describe the mechanism of action of viagra? |
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Definition
| It is a potent inhibitor of Phophodiester enzymes, which hydrolyzes cAMP. So no PDEs, constant cAMP/cGMP and no GMP (constant cellular action) |
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Term
| What are the potential side effects of aberrant signal transduction |
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Definition
| Cause of many diseases including cancer, inflammatory diseases, cardiovascular disease and neuropsychiatric disorders |
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Term
| Does boling blue algae kill it? What are the main issues with blue-gree algae? |
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Definition
| No, it doesn't destroy the toxins. They exist from high levels of phosphates or nitrates (which lead to proliferation of cyanobacteria). |
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Term
| What does Microcystin -LR do? |
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Definition
| Potent inhibitor of phosphoprotein phosphatases - Leads to no futile cycle (haha) but also leads to inability to generate Pi and eventually regenerate ATP |
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Term
| Is microsystin a potent inhibitor? How do you know? |
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Definition
| Yes! Has an I0.5 less than 0.1nM. Also it is now considered a bio terrorism substance |
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