Term
| What would happen to all metabolic processes w/o metabolic control? |
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Definition
| All processes would achieve a state of equilibrium with the external environment (no control of flux) |
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Term
| Process of Annotated Genome Sequencing |
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Definition
Take mapped out genome and identify all ORFs, translate proteins in silico
Take proteins and use gene annotation to assign functions and organize into pathways |
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Term
| Problems associated with "Annotated Genome Sequencing"? |
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Definition
1) up to 50% of sequenced genes encode for unknown proteins
2) don't no about any in vivo conditions
3) mistakes are common |
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Term
| What are the 3 levels of protein function? |
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Definition
Phenotypic - protein's overall function for entire organism
Cellular - protein's location and interactions in cell
Molecular - precise biochemical function of protein |
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Term
| Approximately how much of an organism's genome encodes for proteins? |
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Definition
| Approximately 90% of genome |
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Term
| What are 3 main properties associated with enzymes? |
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Definition
Speed - can increase rate up to 10^14 fold
Specificity - no side reactions; specific S
Control - regulation of flux & activity |
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Term
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Definition
| Any molecule/ion necessary for functioning of the enzyme (no co-factor present, no activity from ENZ) |
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Term
| Two main types of co-factors? |
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Definition
Metal Ions (used by all kinases)
Organic Cofactors/Coenzymes |
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Term
| What is a 'prosthetic group'? |
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Definition
| It is a co-enzyme (organic co-factor) that is covalently attached to an enzyme |
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Term
| Holoenzyme vs. Apoenzyme? |
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Definition
Holoenzyme = enzyme + all co-factors (active)
Apoenzyme = enzyme w/o co-factors (inactive) |
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Term
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Definition
| Any small molecule that REVERSIBLY binds to a protein (includes - substrates, co-factors, etc.) |
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Term
| How many groups are enzymes divided into by commission numbers? What is a commission number? |
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Definition
6 groups in total
Commission # = 4 digit number unique to each enzyme (Class, Subclass, Subclass-Subclass, Substrate) |
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Term
| At a constant T & P, what 2 factors determine how much of a given ligand is bound to an enzyme? |
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Definition
1) Concentration of the ligand
2) Dissociation constant - measures affinity (low constant = high affinity) |
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Term
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Definition
| Enzymes that catalyze IDENTICAL chemical reactions, but are encoded for by different genes |
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Term
| Specific example of isozymes in muscle vs. heart tissue.... |
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Definition
LDH - two different types, two different locations
Muscle LDH - made of 4 M chains; favours pyruvate to lactate transformation during exercise
Heart LDH - made of 4 H chains; favours lactate to pyruvate transformation during recovery from burst muscle work |
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Term
| What did M-M realize about enzyme catalyzed reactions that were unique? |
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Definition
| ENZ reactions saturate with substrate - have a maximal level of activity that the ENZ can perform, even if substrate is increased past this level, no increase in activity |
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Term
| What are the units of Vo and Vmax? |
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Definition
| umol of S->P conversion/minute per mg of ENZ |
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Term
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Definition
| Vo = (Vmax x [S])/(Km + [S]) |
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Term
| What is the most common way to measure S to P conversion in an ENZ catalyzed reaction? |
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Definition
| Spectrophotometer - measure changes in absorbance and use Beer's Law to calculate changes in concentration |
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Term
| What is the equation for kcat? |
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Definition
kcat = Vmax/[ENZ]
Units = s^-1 |
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Term
| What is the Michaelis constant? |
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Definition
| Km = [S] when Vo is at 1/2Vmax (substrate concentration at half maximal activity) |
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Term
| How do you calculate catalytic efficiency? |
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Definition
catalytic efficiency = kcat/Km = Vmax/Km
Because kcat is proportional to Vmax (kcat = Vmax/[ENZ])
UNITS = M^-1 s^-1 |
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Term
| What do we want to maximize in order to increase catalytic efficiency? |
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Definition
| Maximize Vmax and minimize Km (want high activity and high affinity) |
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Term
| What ENZ can undergo non-M-M kinetics? |
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Definition
ONLY enzymes with QUATERNARY STRUCTURE, ENZ need to be multimeric to undergo non-MM kinetics
Non-MM kinetics is much less common than MM-ENZ kinetics, but is still present and important |
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Term
| What kind of Vo vs. [S] plot is seen for ENZ obeying MM kinetics vs. non-MM kinetics? |
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Definition
MM ENZ - see hyperbolic curve; no cooperativity
Non-MM ENZ - see sigmoidal curve; positive cooperativity between subunits |
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Term
| Homotropic vs. Heterotropic ENZ? |
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Definition
Homotropic - allosteric modulator IS the substrate, and therefore acts at the active site
Heterotropic - allosteric modulator binds at some allosteric site other than the active site; modulator is NOT substrate |
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Term
| What are the allosteric effectors of pyruvate kinase? What are each of their effects on the curve? |
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Definition
Activator - G6P - increases PK activity; causes the curve to become hyperbolic (nH = 1) and increases Vo
Inhibitor - ATP - decreases PK activity; shifts curve to the right and decreases Vo |
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Term
| What does the Hill coefficient signify? |
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Definition
Degree of cooperativity between subunits of a protein
nH = 1 -> NO cooperativity; hyperbolic curve
nH > 1 -> positive cooperativity; sigmoidal curve |
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Term
| What is the most common form of inhibition in cells? |
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Definition
| Competitive Inhibition - inhibitor competes with S for binding at active site of free ENZ |
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Term
| What are the effects of competitive vs. mix-competitive inhibition on Vmax and Km? |
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Definition
Competitive - ONLY affects Km (increases Km); no change in Vmax
Mix-Competitive - affects both Km and Vmax (increases Km, decreases Vmax) |
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Term
| In competitive inhibition, why is only Km altered? |
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Definition
| See an increase in Km because interactions between ENZ and S are altered (change in affinity), but no change in ENZ conformation or max rate |
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Term
| What type of inhibition is more frequently encountered with pacemaker enzymes? |
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Definition
| Mix-competitive inhibition at allosteric sites (causes change in Km and Vmax) |
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Term
| For any given inhibitor, what 2 things do you want to know? |
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Definition
1) its pattern of inhibition (mixed comp or comp)
2) its effectiveness/potency (measured by I0.5) |
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Term
| Effects of an ACTIVATOR on Km and Vmax? |
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Definition
Usually, activator only causes a decrease in Km (increased affinity), but can also increase Vmax of ENZ
ALSO, may interfere with interactions between inhibitors and the ENZ |
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Term
| What 3 things do we want to know about any activator molecule? |
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Definition
1) its effect on Vmax and Km values of ENZ
2) its potency (A0.5, Ka value)
3) its effect on interactions between ENZ and I moc. |
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Term
| Example of activator molecule interfering with ENZ and I interactions? |
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Definition
Look at PK in the presence of Glu (mix-comp I) and Asp (activator)...
In the presence of increasing concentrations of Asp, the inhibition by Glu is decreased (Asp interferes with EN-Glu interactions) |
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Term
| General rule about the relationship between T and Vmax? |
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Definition
Generally, as T increases, Vmax increases (as long as ENZ remains in stable native conformation)
Usually, both Km and Vmax are affected by T |
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Term
|
Definition
Q10 = Vo(T+10)/Vo(T)
UNITS = degrees CELSIUS |
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Term
| What is the Q10 value for most mammals (homeotherms)? |
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Definition
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Term
| What temperature ranges do thermophiles and hyperthermophiles live at? |
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Definition
Thermophiles = 90 degrees C
Hyperthermophiles > 90 deg C |
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Term
| What are 3 possible developments that have stabilized the proteins found in extremophiles? |
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Definition
1) Increased number of charged residues to increase # of ionic bonds
2) Increased # of hydrophobic residues; hydrophobic effect is stronger at higher T
3) Decreased # of polar residues; H-bonds are weaker at higher T |
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Term
| What do low Q10 values of poikilotherms indicate? |
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Definition
Poikilotherm = organism whose internal temperature varies; opposite = homeotherms (mammals)
Low Q10 values (closer to 1) shows that metabolic processes are NOT as affected when exposed to low environmental temperatures (leading to lower internal temperatures) |
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Term
| What are thermal kinetic windows? |
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Definition
TKW = temperature range at which an E's maximal affinity for S (lowest Km value) occurs
These TKW's are typically the physiological temperature ranges of the organisms, to optimize ENZ functioning |
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Term
| What is the range of pH optima for most enzymes? |
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Definition
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Term
| Why does Vo change as pH changes? |
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Definition
1) Alters ionization state of residues (changes charges) on ENZ
2) Alters ionization state of residues on SUBSTRATE
3) At extreme pH, causes ENZ denaturation |
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Term
| Why do we eliminate all ENZ contaminants before studying? |
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Definition
- Dont want to interfere with kinetics/structure
- Accurately determine in vivo PTMs
- Allows for antibody production against pure ENZ
- Use aa sequencing to figure out encoding genes |
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Term
| What is the "Humpty Dumpty" Principle? |
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Definition
| That the whole is greater than the sum of its parts; essentially, in vitro cell components does NOT EQUAL in vivo cell intact |
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Term
| [ENZ] in vitro vs. in vivo cell? |
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Definition
[ENZ] in vitro <<<< in vivo
in vitro = 1-10 ug/mL
in vivo = 100-200 mg/mL |
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Term
| What is the dilution problem? |
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Definition
| In vitro [ENZ] is so low that subunits dissociate from one another and ENZ becomes non-functional |
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Term
| What are two solutions to the dilution problem? |
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Definition
1) Do large scale preps to keep [ENZ] high
2) Add some sort of aggregating agent into mix (e.g. 20% glycerol) |
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Term
| When comparing mass action ratio and Keq, when can you tell the reaction is probably catalyzed by a pacemaker enzyme? |
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Definition
| If K >>> mass/action, then reaction is far shifted from equilibrium (highly negative free energy) |
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Term
| What are the majority of chemical reactions in any metabolic pathway? |
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Definition
| Equilibrium reactions (where K = mass/action ratio, approximately) |
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Term
| Where are pacemaker enzymes usually located in pathways (circumstantial evidence)? |
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Definition
| Usually located at 1st committed step OR at major branchpoints |
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Term
| What is metabolic control analysis used for? |
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Definition
| Tries to quantify relative contribution of each ENZ in pathway to management of flux in intact system |
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Term
| What are the 2 predictions of MCA? |
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Definition
1) Control of flux is shared by many, if not all ENZ in pathway
2) Degree of control by each ENZ can change as physiological status changes |
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Term
| Two ways to go about MCA? |
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Definition
Change flux of pathway - must be done in vivo, hard to do
Change in activity of ENZ - usually through genetic engineering |
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Term
| What is the flux control coefficient? |
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Definition
CJE = change in flux/change in activity of enz
Ranges from 0-1; if =1, then ENZ is very involved in pathway flux |
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Term
| How is energy gathered from lipids and carbohydrates within the cell? |
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Definition
| Covalent bonds are oxidized to release energy |
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Term
| How many high energy bonds exist in ATP? |
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Definition
| Have 2 high energy phosphoanhydride bonds |
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|
Term
| What is required as a co-factor in ATP hydrolysis? |
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Definition
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|
Term
| Function of nucleoside diphosphate kinase? |
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Definition
| Converts ATP + NDP -> ADP + NTP (can get any other nucleoside triphoshate from ATP) |
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Term
| In mammals, why does inorganic PPiase used? |
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Definition
| To give thermodynamic push to drive macromolecule synthesis (gets rid of PPi, to push equation to the right, so continue biosynthesis) |
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Term
|
Definition
| Have a PPi dependent PFK in plants to make use of PPi during energy depletion (have no PPiase) |
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Term
| Why use ATP instead of PPi in mammals, even though PPi likely evolved first? |
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Definition
| ATP - generates more energy per molecule; ATP and other NTPs are more chemically versatile |
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Term
| 3 main roles of catabolism: |
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Definition
1) Generate ATP
2) Generate "reducing power" (e- for ETC for ATP generation)
3) Generate anabolic precursors/intermediates |
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Term
| What are the most important e- carriers in redox reactions in cells? |
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Definition
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|
Term
| Function of NADP/NADPH system vs. NAD/NADH system? |
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Definition
NADP/NADPH - shuttle electrons from oxidative catabolic paths to reductive anabolic paths
NAD/NADH - shuttle electrons from oxidative catabolic paths to ETC |
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Term
| Are catabolic reactions oxidative or reductive? Anabolic? |
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Definition
Catabolic = oxidative (eg. kreb's cycle & glycolysis)
Anabolic = reductive |
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Term
| In amphibolic pathways, how can flux in one direction be controlled without altering flux in the other direction? |
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Definition
| Always have 1 pacemaker ENZ unique to each path; usually 1 has to couple catalysis with ATP hydrolysis to make sure both stay exergonic |
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Term
| How can you prevent futile cycles in amphibolic pathways? |
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Definition
Temporal separation - same regulatory signal has opposite effects on the two ENZ (activates one, inhibits other)
Spatial segregation - compartmentalize different pathways |
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Term
| Where does fatty acid degradation vs. biosynthesis occur in mammals? |
|
Definition
Degradation = mitochondria
Synthesis = cytosol |
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Term
| Where does fatty acid degradation vs biosynthesis occur in plants? |
|
Definition
Degradation = glyoxysome
Synthesis = plastid (chloroplast) |
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|
Term
| What is the primary function of cyclic pathways? |
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Definition
| To link catabolic & anabolic pathways (e.g. TCA cycle) |
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Term
| Two examples of anaplerotic carboxylases? Functions? |
|
Definition
Pyruvate carboxylase (animals), PEP carboxylase (plants)
Maintain high [oxaloacetate], keep intermediates for TCA cycle abundant |
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Term
| Why are two reasons it is incorrect to assume that mRNA is a very good quantitative indicator of ENZ activity? |
|
Definition
1) miRNA - binds to mRNA and silences genes by blocking translation
2) Riboswitches - allosteric mRNA that controls translation; ENZ w/ riboswitch regulates its own activity |
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Term
| What residues are tagged by UB? |
|
Definition
|
|
Term
|
Definition
| E1 activase, E2 conjugase, E3 ligase |
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|
Term
| What destroys proteins targeted by poly-UB? |
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Definition
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|
Term
| What human diseases may be associated with poly-UB? |
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Definition
| Neurodegenerative (Hunt, Alz); linked to accumulation of protein granules, defect in UB-proteasome machinery |
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Term
| 6 methods of fine control? |
|
Definition
Change Substrate
Change pH
Change allosteric effector concentrations
Reversible covalent mods
Subunit association/dissociation
Metabolon formation |
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Term
| What is the relationship between changing [S] and rate of reaction in fine MC? |
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Definition
| 2-3x increase in [S], can increase rate up to 100 fold |
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|
Term
| Example of change in pH as method of fine MC? |
|
Definition
During light/dark reactions in plants...
When going from dark to light, pH increases from 7 to 8 (7 in dark, 8 in light)
Pump H+ out to increase pH; this increase in pH activates ENZ in Calvin cycle (light reactions) |
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Term
| Type of control for activators vs. inhibitors? |
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Definition
Activators = feedforward control
Inhibitors = feedback control |
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|
Term
| What allows for AMP amplification? |
|
Definition
ADENYLATE KINASE (catalyzes ATP + AMP <-> 2 ADP
Small change in ATP causes large change in AMP |
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Term
| When looking at glycolysis and F-6-P pathway, effectors of PFK vs. FBPase |
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Definition
PFK - AMP (activator), ATP (inhibitor)
FBPase - AMP (inhibitor)
When low ATP, AMP levels rise to favor flux through path |
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Term
| Only 3 residues that can be phosphorylated? |
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Definition
| Serine, Threonine, Tyrosine |
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|
Term
|
Definition
| Intracellular E monitor; shuts off anabolic pathways that use ATP and activates catabolic pathways that make it |
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Term
| Activity of glycogen phosphorylase/synthase in exercising vs. resting muscle? |
|
Definition
Exercising - activate phosphorylase, inhibit synthase
Resting - inhibit phosphorylase, activate synthase |
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Term
| Disulfide-thiol interconversion in light & dark reactions? |
|
Definition
| FD and TR act as e- carriers to reduce the disulfide into a dithiol when light -> activates ENZ to increase reducing power by 50x |
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Term
| PEPCase activity in castor oil seeds? |
|
Definition
Germinating - mono-UB is inhibitory and increases Km
Developing - phosphorylation activates PEPCase |
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|
Term
| Examples of subunit association/dissociation as a means of fine MC... |
|
Definition
1) A-CoA-Case - deP it is in a polymer form (active), in the fed state; P it is in a monomer form (inactive), in starved state
2) PK - when [activator] is high, in homotetrameric form; when [activator] low, in monomeric form (inactive); in pituitary gland, can only bind TH when in monomers |
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|
Term
|
Definition
| ENZ that reversibly binds to particulate structures (membrane, cytoskeleton) in the cell, depending on the cell's metabolic status |
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|
Term
| 3 major advantages of metabolons? |
|
Definition
1) Close proximity of active sites increases efficiency (can alter kcat of enzyme)
2) Metabolite channeling - prevents futile cycles
3) Doesn't tax solvent capacity of cell |
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|
Term
| What ENZ degrades cAMP -> AMP? |
|
Definition
|
|
Term
|
Definition
| Adenylyl cyclase (AMP + Pi -> cAMP) |
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|
Term
| What is the primary target of cAMP? |
|
Definition
| PK-A (cAMP binding causes subunits to dissociate and become active!) |
|
|
Term
| Main differences between cAMP and AMP? |
|
Definition
AMP has NO effect on PK-A (instead acts on AMPK)
AMP signals E deficiency in the cell (based on changes in ATP levels)
[cAMP]<<<<[AMP]
[cAMP] = 1-10 uM
[AMP] = 100-500 uM; 0.1-0.5 mM |
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|
Term
| What are the typical concentrations of [Ca] in cells normall vs. when 2nd messenger cascade is activated? |
|
Definition
Normally [Ca] < 1 uM
After activation of cascade [Ca] = 1-10 uM |
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|
Term
| How many binding sites are located on calmodulin? |
|
Definition
|
|
Term
| Relative # of CBP's in plants vs. animals? |
|
Definition
| Plants have over 50, animals only have 1 isoform of CaM |
|
|
Term
| What is true of almost all 2nd messengers? |
|
Definition
| They have their own protein kinase (2nd messenger cascades rely heavily on phosphorylation) |
|
|
Term
| How much of an increase in signal amplifications do 2nd messenger cascades allow for? |
|
Definition
|
|
Term
| PL-C, DAG, IP3, Ca2+ cascade.. |
|
Definition
Upon external signal, PL-C hydrolyzes GTP and cleaves a membrane phospholipid into DAG and IP3
DAG binds to PK-C and IP3 opens Ca2+ channels to increase Ca2+ levels in cell
PK-C requires both Ca2+ and DAG binding to activate |
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