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83 Cards in this Set
- Front
- Back
What did early work of enzyme kinetics involve?
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crude preps, only observe reaction of reactants to products
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What eventually were we able to do with enzyme kinetics?
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Measure rates
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In the presence of ________ and _________, enzyme kinetics became a powerful tool.
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Competitors and Inhibitors
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For chemical kinetics where A->P, at constant T what is rate?
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Frequency of A colliding
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What is proportionality constant for chemical kinetics?
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Rate constant k
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In chemical kinetics A -> P, what is instantaneous appearance of P or disappearance of A called?
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velocity v; v=d[P]/dt = -d[A]/dt = k[A]
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What are units of velocity and rate constant?
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Velocity is M/sec; rate constant is 1/sec
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What does order of reaction correspond to?
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Molecularity - number of reactants that must collide to form P
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1ST ORDER IS WHAT TYPE OF MOLECULARITY?
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Unimolecular reaction
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What is velocity and order nad molecularity of 2A -> P?
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bimolecular 2nd order; v = d[A]/dt = k[A]^2
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What are units for rate constant k in second order reaction?
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M^-1sec^-1
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What is v or instantaneous velocity for A + B -> P?
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k[A][B]
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How do we know that A + B -> P is second order?
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Because first order in A and first order in B = 2nd order
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Why are higher order reaction such as termolecular uncommon?
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Because must rely on simultaneous collision of 3 or more molecules
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What does rate equation describe?
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Progress as a function of time
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What is rate equation for 1st order reaction A -> P?
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ln[A] = ln[A]0 - kt
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What is the graph look like for rate equation of 1st order reaction such as A-> P?
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plot of ln[A] as y axis and time as x axis and straight line with slope -k
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For first order reactions such as A-> P, time for half A to decompose is _______.
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Half life
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Half life of A in first order reaction A->P is constant and independent of ________.
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[A]o
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What is the formula for half life in first order reaction?
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t1/2 = .693/k
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What is reaction equation for second order reaction with 1 type reactant? so 2A - > P
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1/[A] = 1/[A]o + kt
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What is half life of second order single type reactant dependent on?
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[A]o
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What is the equation for half life of second order single type reactant?
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t1/2 = 1/ k[A]o
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How do we distinguish first order from second order reaction plot?
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1st order - ln[A] vs. t
2nd order - 1/[A] vs. t |
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For second order reaction: A + B -> P in which B concentration much greater than A what does rate depend on? Why?
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Concentration of A since in limited supply
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E + S ⇄ ES → P + E, When S is high enough to convert all E to ES, what is rate limiting step?
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Second Step
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E + S ⇄ ES → P + E, When S is high enough to convert all E to ES, the overall reaction is still sensitive to S concentration. T/F
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False, it is insensitive to S concentration
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E + S ⇄ ES → P + E, When S is high enough to convert all E to ES. Which steps are reversible and irreversible?
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K1 reversible and K2 irreversible
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What is Michaelis – Menten Equation: with respect to E + S ⇄ ES → P + E?
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It describes rate of enzymatic reaction as function of S
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What is rate of P formation in E + S ⇄ ES → P + E?
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v = k2[ES]
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What is rate of production of ES in E + S ⇄ ES → P + E?
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Rate of production of ES is Δ between rates of appearance and disappearance
d[ES]/dt = k1[E][S] – k-1[ES] – k2[ES] |
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What are the two assumptions necessary to integrate the Michaelis-Menten Equation with E + S ⇄ ES → P + E?
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1. Assumption of equilibrium in that k-1 » k2 so first step reaches equilibrium Ks = k-1/k1 = [E][S]/[ES] where ks is dissociation constant of 1st step
2. Assumption of steady state: under common physiological conditions [S] » [E]; with exception of first millisec [E] and [ES] remain ≈ constant until [S] exhausted |
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In E + S ⇄ ES → P + E, ES maintains what state and how is it treated?
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Steady state and constant value
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Who first proposed the steady state assumption?
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GE Briggs and JBS Haldane
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How are kinetic expressions useful?
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Experimentally measurable
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_________ and __________ are not measurable in E + S ⇄ ES → P + E. But ___ is measurable.
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ES and E; Et = ES + E
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E + S ⇄ ES → P + E; What is rate equation for overall reaction as function of E and S?
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k1[E][S] = k-1[ES] + k2[ES]
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E + S ⇄ ES → P + E; How can you rearrange this equation to use only K's and give Michaelis Constant Km?
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Km = (k-1 +k2)/k1 or KM[ES] = ([E]T – [ES])/[S]
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In Michaelis Menten Equation, using vo rather than v minimizes some complications such as ....
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Reversible reactions
Product inhibition of E Progressive inactivation of E |
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How do we meet steady state conditions in Michaelis Menten Equation for E + S ⇄ ES → P + E?
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Concentration of S has to be greater than E so that E can repeatedly bind to S and convert to P thus ES is constant
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What is maximum velocity in E + S ⇄ ES → P + E?
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It is when there is increase in S concentration so E is saturated all in ES form. Vmax = k2[E]
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Combining all of the steps what is the full equation (End equation) for Michaelis Mentin Equation?
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v0 = Vmax[S]/(Km + [S])
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When does Km = [S]?
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When Vo = 1/2Vmax
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What is Km?
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It is S concentration term and measure of catalytic efficiency.
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What does small Km and large Km mean?
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Small Km means Vmax at lower [S] or higher catalytic efficiency; large Km means Vmax at higher [S] or lower catalytic efficiency
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T/F Km is the same for every enzyme substrate pair.
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False it is unique for everyone enzyme substrate pair.
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Km is also a function of ________ and ________ while being unique for every enzyme substrate pair.
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Temperature and pH
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What is Ks= ? and what does it mean?
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k-1/k1 ; it is the dissociation constant of Km
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What happens when Ks increases or decreases?
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As Ks increases E's affinity for S decreases and As Ks decreases, E's affinity for S increases
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Km can be a measure for E's affinity for S. If Km high ... and if Km low...
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High Km - low affinity for S
Low Km - high affinity for S |
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What is the catalytic constant or turnover number?
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Number of reactions/active site/ unit of time
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What is the formula for the catalytic constant?
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Kcat = Vmax/[E]t
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What does it mean if Kcat is high and if Kcat is low?
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Kcat high - fast turnovers, fast enzyme
Kcat low - low turnovers, slow enzyme reaction |
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Even thought Kcat is constant, Vmax is approximately equal to ____.
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[E]t
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In a simple E equation Kcat = ?
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k2
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What happens when [S] << Km
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Little ES forms and [E] = [E]t
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In determining Vmax in Vo vs. [S] plot, what is Vo when [S] = 10Km? Also why is it difficult to determeine Vmax from this plot?
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Vo = about 91% of Vmax
Vmax is often underestimated by this plot |
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What did Hans Lineweaver and Dean Burk formulate?
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They formulated a better way of calculating Vmax by using reciprocal of Michaelis Menten equation. -> 1/Vo = (Km/Vmax)(1/[S]) + (1/Vmax)
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What is a disadvantage of Lineweaver Burk Plot?
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There is a crowding of data along vertical axis
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What do small errors in [S] and Vo lead to in Lineweaver Burk Plot Data collection?
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Large errors in 1/Vo -> large errors in Km and Vmax
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T/F Steady state kinetics can establish reaction mechanisms.
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False
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What happens if kinetic data does not match given mechanism?
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Mechanism must be rejected
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What kind of insight to steady state kinetics provide for ES?
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Provides insight into buildup and breakdown of ES, provides little information on nature of ES, Can not provide information on number of intermediates of ES
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What are bisubstrate reactions and how much of chemical reactions to they make?
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60% of chemical reactions are bisubstrate reactions; bisubstrate - 2 substrates and 2 products
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What kind of reactions are bisubstrate ones usually?
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Transfer Reactions or Oxidation Reduction Reactions
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What are two types of Bisubstrate reactions?
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Sequential Reactions - all S’s bind with E before reaction occurs and P’s released
Ping Pong reactions: group transfer reactions where 1 or more P’s released before all S’s bound |
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What is the ordered mechanism of a sequential reaction?
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Binding of E to S required before it can open second binding site.
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What is random mechanism to sequential reactions?
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No specific order of S binding , both binding sites are available
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What are Enzyme inhibitors?
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They can alter Enzyme activity by altering S binding or P release
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What are irreversible inhibitors?
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They bind so tightly that they block enzyme activity completely
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What is competitive inhibition?
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Ihibition where I competes for E binding site.
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Who do competitive inhibitors compete with for E binding site?
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S they usually look like S as well
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What is one of the physiological methods of E control?
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Competitive inhibition by Product P
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How do Transition state analogs function as good inhibitors?
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They mimic TS
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What does degree of competitive inhibition depend on?
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Fraction of E that was bound to Inhibitor
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What is an uncompetitive inhibitor?
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Where I binds directly to ES complex and not E; doesn't need to resemble S; distorts E active site rendering it catalytically inactive
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What is mixed inhibition?
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Inhibitors bind both E and ES complex; may be reversible, affect S binding, affect catalytic activity, Binds to E sites participating in both catalysis and S binding
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What is an example of a good mixed inhibitor?
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Metal Ions
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What is another name of mixed inhibitors?
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Noncompetitive inhibitors
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How do organisms regulate and coordinate metabolism?
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Control enzyme availability - amount of E depends on synthesis and degradation; and enzyme activity - controlled by product inhibition - feed back
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What is allosteric control of enzyme activity?
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Binding at site other than active site to change conformation
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What is covalent modification of E?
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Phosphorylation/Dephosphorylation of E; catalyzed by protein kinases and protein phosphates
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_______ of human protein participating in biological processes is subject to covalent modification,
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30%
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