Lecture 5 - Protein Function Flashcards by Madeline Squires

oxygen binding to myoglobin and hemoglobin:

oxygen (O2) binds to myoglobin and hemoglobin via ___, a specialized oxygen-binding ____

heme, a specialized oxygen-binding cofactor

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oxygen binding to myoglobin and hemoglobin:

oxygen requires a ___-___ ____ for effective binding

protein-bound metal for effective binding

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oxygen binding to myoglobin and hemoglobin:

myoglobin:

function: ___ oxygen in muscle cells for later use

structure: ____ (___ ___ ___)

stores oxygen in muscle cells for later use

monomeric (single protein chain)

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oxygen binding to myoglobin and hemoglobin:

hemoglobin:

function: ___ oxygen in the bloodstream from the lungs to tissues

structure: ____ (___ ___, each with a ___ ___)

cooperativity in binding: oxygen binding to one subunit ____ ____ in the others

transports oxygen in the bloodstream from the lungs to tissues

tetrameric (4 subunits, each with a heme group)

increases affinity in the others

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oxygen binding to myoglobin and hemoglobin:

heme structure:

___-containing ____ ring that binds ____

___ (__) in heme binds ____ (___), forming an ____ complex

iron-containing porphyrin ring that binds oxygen

iron (Fe2+) in heme binds oxygen (O2), forming an oxygenated complex

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oxygen binding to myoglobin and hemoglobin:

oxygen binds to ___ in heme and is stabilized by ___ ___ and ___ ___ residues

this prevents oxidation of ___ to ___, which would make oxygen binding ____

Fe2+ in heme and is stabilized by His E7 and His F8 residues

Fe2+ to Fe3+, which would make oxygen binding irreversible

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oxygen binding to myoglobin and hemoglobin:

protein side chains alone lack ____ for / cannot ___ oxygen effectively

affinity for / cannot bind oxygen effectively

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oxygen binding to myoglobin and hemoglobin:

____ bind O2 well, but generate __ ___ in solution and could be ____ (__ to ___)

metals bind O2 well, but generate free radicals in solution and could be oxidized (Fe2+ to Fe3+)

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oxygen binding to myoglobin and hemoglobin:

the solution is to capture O2 molecule with ___ in ____ (____ ____ or ____ ____)

heme ___ ___ within the protein environment

myoglobin uses a ___ ___; hemoglobin has __ ___ ___ for ___ ___

heme in protein (myoglobin monomer or hemoglobin tetramer)

shields Fe2+ within the protein environment

single heme; hemoglobin has 4 heme groups for cooperative binding

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single site binding to myoglobin:

binding of O2 monitored by ___ ____:

deoxyhemoglobin and deoxymyoglobin (no O2 bound) appear ___ ; found in ____

oxyhemoglobin and oxymyoglobin (O2 bound) appear ___; found in ____

UV-Vis spectroscopy

purple; found in veins

red; found in arteries

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single site binding to myoglobin:

myoglobin binds O2 ____ (would be) bad for ___ in blood vessels bc no ___ occurs, but great for ____

tightly (would be) bad for transport in blood vessels bc no release occurs, but great for storage

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single site binding to myoglobin:

____ binding to hemoglobin ____ makes it a better ____ protein

cooperative binding to hemoglobin tetramer makes it a better transport protein

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single site binding to myoglobin:

myglobin (___ ___):

binds O2 ___

___ binding curve (n=__), meaning O2 binding is ____

always holds onto ___, even at ____ pressures

(single subunit):

tightly

hyperbolic binding curve (n=1), meaning O2 binding is independent

oxygen, even at lower pressures

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single site binding to myoglobin:

hemoglobin (____)

___ O2 binding (___ curve, n = ___)

releases O2 at ___ ____, making it better for ___

releases O2 where needed (___ at ___ pressure)

binds O2 in ____ (___ pressure)

(tetramer)

cooperative O2 binding (sigmoidal curve, n=4)

lower pressures, making it better for transport

(tissues at venous pressure)

lungs (arterial pressure)

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single site binding to myoglobin:

Kd = [___]^__

[S 0.5]^n

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hemoglobin: allosteric cooperativity changes:

____ state (low affinity)

____ state (high affinity)

change in affinity helps to pick up ___ in ___ and drop it off in ____

tense state (low affinity)

relaxed state (high affinity)

O2 in lungs and drop it off in tissues

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hemoglobin: allosteric cooperativity changes:

the first O2 binds _____ to hemoglobin, initiating a ___ to ___ ____

non-cooperatively to hemoglobin, initiating a T to R transition

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hemoglobin: allosteric cooperativity changes:

the next 3 O2 molecules bind ____

this means that each successive binding ____ hemoglobin’s ___ for oxygen

increases hemoglobin’s affinity for oxygen

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hemoglobin: allosteric cooperativity changes:

the transition from T to R (and back) occurs when ___ to ___ O2 molecules are bound

1 to 3 O2 molecules are bound

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hemoglobin: allosteric cooperativity changes:

in revers, the ____ ___ triggers the start of R to T change

first unbinding triggers the start of R to T change

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hemoglobin: allosteric cooperativity changes:

the T state (___ affinity) is dominant at ___ oxygen pressures (e.g. in ____)

the R state (___ affinity) dominates at ___ oxygen pressures (e.g. in ___)

___ shape of the curve indicates ___ binding

(low affinity) is dominant at lower oxygen pressures (e.g. in tissues)

(high affinity) dominates at higher oxygen pressures (e.g. in lungs)

sigmoidal shape of the curve indicates cooperative binding

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hemoglobin: allosteric cooperativity changes:

on the hill plot, the low-affinity state (T, nH = __) and high-affinity state (R, nH = ___)

(T, nH = 1) and high-affinity state (R, nH = 3)

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hemoglobin: allosteric cooperativity changes:

the slope change reflects ____, showing how O2 binding at one site ___ other sites

cooperativity, showing how O2 binding at one site influences other sites

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hemoglobin: allosteric cooperativity changes:

hemoglobin exemplifies _____

binding of O2 at one site affects ___ ____ at distant (___) sites

this cooperative mechanism ensures efficient O2 uptake in the ____ and release in ____

allostery

O2 binding at distant (protein) sites

lungs and release in tissues

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Physical Interactions Leading to T to R Change: oxygen binding alters ___ of ___ ___, exerting force on ___ ___

pucker of heme ring, exerting force on helix F

Physical Interactions Leading to T to R Change: conformational change from T to R involves breaking ___ ___ between the ___-____ interface

ion pairs between the a1-B2 interface

Physical Interactions Leading to T to R Change: structural changes are ____ ___ ____ stabilizes ____ ___, and ___ ____ stabilizes ___ ____

O2 binding stabilizes R state, and R state stabilizes O2 binding

Physical Interactions Leading to T to R Change: in the T state (low oxygen affinity): Fe2+ is in a ___-___ state with ___ ___ electrons the iron sits slightly ____ of the ___ of the heme ring when O2 binds, Fe2+ transitions to a ___-___ state (__ ___ electrons) this causes the Fe2+ to move ___ the ___ of the heme ring, which pulls on ___ ____ attached to ___ ___

high-spin state with 4 unpaired electrons out of the plane of the heme ring low-spin state (no unpaired electrons) into the plane of the heme ring, which pulls on his F8 attached to helix F

Physical Interactions Leading to T to R Change: the movement of Fe2+ and the flattening of the heme ring cause ___ ___ to ____ this shift ___ key ___ ___ at the ___-____ interface, triggering a ___ structural transition from the ___ state to the ___ state

helix F to shift breaks key ion pairs at the a1-B2 interface, triggering a global structural transition from the T state to the R state

Physical Interactions Leading to T to R Change: tissues are at pH ___, and this ___ the affinity for O2 lungs are at pH ___, and this helps to ___ the affinity for O2

7.2, and this lowers the affinity for O2 7.6, and this helps to raise the affinity for O2

Catalyzing a Reaction: reaction rates are governed by the ___ of the ___ ___ ____ reaction rates can be increased by ____ the ____ ____

height of the activation energy barrier lowering the activation barrier

enzyme catalysis: enzyme effects: ___ reaction rates operate at ____ reaction conditions (___ temp, pH ~___, ___ ____ concentrations greater reaction ____ (___ catalyze specific reactions, minimizing unwanted ___ ___) capacity for ____ (enzymes can be ____ by molecules, creating ___ ___)

higher reaction rates milder reaction conditions (room temp, pH ~7, low reactant concentrations) specificity (selectively catalyze specific reactions, minimizing unwanted side products) regulation (enzymes can be modulated by molecules, creating feedback loops)

enzyme catalysis: phosphatases accelerate the rate of ___ ____ ____ by 10^__ ___ ____ is one of the fastest enzymes known, catalyzing reactions at the maximum possible rate

phosphate monoester hydrolysis by 10^7 carbonic anhydrase

catalysis by binding the transition state: enzymes lower activation energy and accelerate reactions most effectively by binding to the ___ ___ rather than the ____ itself

transition state, rather than the substrate itself

catalysis by binding the transition state: without an enzyme, the activation energy is ____, making the reaction ___

high, making the reaction slower

catalysis by binding the transition state: enzyme complementary to substrate: if an enzyme binds too tightly to the substrate, it actually ____ the substrate, making it ___ to reach the transition state the enzyme-substrate complex is at a ___ energy state but the transition state still requires ___ activation energy

stabilizes the substrate, making it harder to reach the transition state lower energy state high activation energy

catalysis by binding the transition state: enzyme complementary to transition state: the most effective enzymes work by binding most strongly to the ___ ___, not the ___ the enzyme either ___ ___, or ____ the ___ ___ this lowers the ___ ___, ___ the reaction rate

transition state, not the substrate induces strain, or stabilizes the transition state activation energy, increasing the reaction rate

catalysis by binding the transition state: enzyme pockets lower activation energy by ___ binding to the ___ ___

specifically binding to the transition state

catalysis by proximity: enzymes can accelerate reactions by paying the ____ penalty of bringing two ___ ____ and ___ ___ in the right ____

entropy penalty of bringing two reactants together and arranging molecules in the right orientation

catalysis by proximity: enzymatic example: DNA polymerase is an enzyme that ___ ___ by adding ____ to a ___ ___ DNA polymerase uses ___ ___ and ___ ___ help position the ____ and stabilize ___ ___ the reactants are held in ___ ___, ensuring efficient ___ this speeds up the reaction by reducing the ___ of ___ ___

synthesizes DNA by adding nucleotides to a growing strand magnesium ions (Mg 2+) and aspartate residues help position the nucleotide and stabilize negative charges close proximity, ensuring efficient catalysis

catalysis by specific functional groups: sometimes enzymes merely bring ____ ___ and stabilize the ___ ____ so other molecules (___) can ____

reactants together and stabilize the transition state so other molecules (H2O) can react

catalysis by specific functional groups: in many cases, specific ____ ___ (___ ___ or ___) participate in catalysis by acting as ___, ____, ____, or ____

functional groups (amino acids or metals) participate in catalysis by acting as acids, bases, nucleophiles, or oxidants)

serine proteases: proteases ____ specific sequences by ___ ___ ___ in the ____ around the ____ _____ binding also sets up the bond for ___ ____ by ___ ___

cleave specific sequences by binding amino acids in the substrate around the scissile bond nucleophilic attack by Ser oxygen

serine proteases: all serine proteases share an ____, ____, ____ ___ ___

Asp, His, Ser catalytic triad

serine proteases: serine proteases are a family of ___ that cleave ___ ___ in ____ using a ___ ___ consisting of ____, ____, and ____

enzymes that cleave peptide bonds in proteins using a catalytic triad consisting of serine, histidine, aspartate

serine proteases: serine proteases have an active site containing 3 key residues: Serine (ser): acts as a ____ that attacks the ___ ____ histidine (his): helps ____ Ser by ____ a ____, making it more ____ aspartate (asp): ____ His by ___ ___, enhancing its ability to ___ ____ these residues work together to ___ ___ ___ efficiently

nucleophile that attacks the peptide bond activate ser by accepting a proton, making it more reactive stabilizes His by hydrogen bonding, enhancing its ability to activate ser cleave peptide bonds efficiently

serine proteases: 1. the enzyme binds the ____ ____ at a specific site near the ___ ___ (the ____ to be ___) the ___ ____ determines ____ (different proteases prefer different ___ ___ at the cleavage site)

protein substrate at a specific site near the scissile bond (the bond to be cleaved) binding pocket determines specificity (different proteases prefer different amino acids at the cleavage site)

serine proteases: 2. serine's ____ attacks the ___ ____ of the ____ ____ the forms a ___ ____, ____ the transition state

oxygen attacks the carbonyl carbon of the peptide bond tetrahedral intermediate, stabilizing the transition state

serine proteases: 3. the peptide bond is ____, and one fragment ___ while the enzyme ___ for the ___ ___

broken, and one fragment leaves while the enzyme resets for the next reaction

serine proteases: trypsin recognizes ___ ___ residues like ____ it contains an ____ residue that helps stabilize the ___ ____ substrate chymotrypsin recognizes ___, ____ residues like ____ it has a ___ binding pocket that fits ___ ___ side chains

positively charged residues like lysine asp residue that helps stabilize the positively charged substrate large, hydrophobic residues like phenylalanine hydrophobic binding pocket that fits bulky aromatic side chains

serine mechanism: the ___ ___ acts as a general ___/___ to activate ____ and ____ for ___ ___ and ___ ___ ___

catalytic His acts as a general acid/base to activate Ser and H2O for nucleophillic attack and protonate leaving groups

transition state stabilization: ser proteases stabilize the two tetrahedral intermediates formed during the cleavage mechanism with ___ ___ ___ ____ the tetrahedral intermediate can be observed directly in ___ ____ ____ obtained at ___ pH, where the ___ and ____ are ____ stable

amide backbone electrostatic interactions x-ray crystal structures obtained at high pH, where the oxyanion and hydroxide are more stable

transition state stabilization: the key mechanism responsible for stabilizing the tetrahedral intermediate formed during peptide bond cleavage is the ____ ____, which helps ____ the ___ ___ and ____ up the rxn

oxyanion hole, which helps lower the activation energy and speed up the rxn

transition state stabilization: the tetrahedral intermediate that forms when the nucleophilic oxygen from ser195 attacks the carbonyl carbon of the peptide bond is highly _____ due to a ____ charged ____ (__) that needs ___

unstable due to a negatively charged oxyanion (O-) that needs stabilization

transition state stabilization: the transition state is stabilized by the ___ ___, which is a ___ ___ within the enzyme that ___ the ___ ___ ____ in the transition state this is done through ___ ___ from ____ ___ ___ of ____ and ____ these hydrogen bonds ____ the ___ ___, making the intermediate more ____ and reducing the ____ required for ___ ____ electrostatic interactions: the ____ ___ and nearby ___ help stabilize the ___ ____ this allows the enzyme to ___ the ____ in an ___ ___ for ____ catalysis

oxyanion hole, which is a structural pocket within the enzyme that stabilizes the negatively charged oxyanion in the transition state hydrogen bonding from backbone amide groups of Gly193 and Ser195 neutralize the negative charge, making the intermediate more stable and reducing the energy required for bond cleavage amide backbone and nearby residues help stabilize the charge distribution hold the substrate in an ideal position for efficient catalysis

transition state stabilization: serine proteases stabilize the tetrahedral intermediate using the ____ ____ the oxyanion hole provides ___ ___ that ___ the ___ ___ on the ____ ___ ___ ___ at ___ pH allows direct observation of the ___ ____ this stabilization ___ the ___ ___, making ___ ___ ___ more efficient

oxyanion hole hydrogen bonds that neutralize the negative charge on the transition state x-ray crystallography at high pH allows direct observation of the tetrahedral intermediate lowers the activation energy, making peptide bond cleavage more efficient

determining reaction rates at steady state: E (___) binds to S (___), forming the ___-____ ___ (___) the ES complex can either: - ____ back into ___ + ____ (rate constant ___) - convert into ____ (__) (rate constant ___) the rate of product formation depends on ___ ____ and ____

(enzyme) binds to S (substrate), forming the enzyme-substrate complex (ES) - dissociate back into E+S (rate constant K-1) - convert into product (P) (rate constant K2) ES formation and breakdown

determining reaction rates at steady state: the steady-state phase occurs when the ____ of the ___-___ ___ [__] remains nearly ___

concentration of the enzyme-substrate complex [ES] remains nearly constant

determining reaction rates at steady state: V0 (initial velocity): the reaction rate at the ___ of ___ ___, before ___ ____ V0 is represented by the ___ of the ___ vs. ___ curve

beginning of stead state, before substrate depletion slope of the [P] vs. time curve

determining reaction rates at steady state: Lehninger graph: shows __ ___ at different ___ ___ higher [S] results in: - ____ product formation - ____ initial velocity (V0) when [S] = Km, the enzyme is working at ___ of its max velocity (___/___)

product formation at different substrate concentrations - faster product formation - greater initial velocity (V0) half of its max velocity (Vmax/2)

determining reaction rates at steady state: steady state occurs when ____ remains ___ initial velocity (V0) is measured before ___ ___ higher substrate concentrations ____ Vo

[ES] remains constant substrate depletion increase V0

determining reaction rates at steady state: reactions are characterized by "___ ___" (__) during at ___ ___ (after __ __, before ___ is ___)

"initial velocity" (V0) during at steady state (after induction period, before substrate is depleted)

Michaelis-Menten/Briggs-Haldane Model: key parameters: Km - how well does the ___ bind ___? k2 - how well does the ___ convert ___ to ____

enzyme bind S enzyme convert S to P

Michaelis-Menten/Briggs-Haldane Model: if Vmax can be identified from asymptote, can identify ___ and ___ by ___ if Vmax cannot be identified from asymptote, curve can be fit to ___ ___

Km and k2 by inspection V0 equation

Inhibitors: Competitive: inhibitor (I) competes with the substrate (S) for the ___ ____ if the inhibitor binds, the substrate ____ ____, ___ the reaction can be overcome by ____ ___ (since more ___ competes for ___) ___ Km (___ affinity for S) because ___ substrate is needed to reach ____/___) Vmax remains ____ because ____ ___ can outcompete the ____

active site cannot bind, blocking the reaction increasing [S] (since more substrate competes for binding) increases Km (lower affinity for S) because more substrate is needed to reach Vmax/2 unchanged because high [S] can outcompete the inhibitor

Inhibitors: Non-competitive: the inhibitor binds to an ____ site, not the ___ site substrate can still ____, but ___ ____ is impaired inhibition cannot be overcome by ___ ___ because the inhibitor affects ___ ___, not ____ Vmax ____ because the enzyme is rendered ____ ____ Km ___ the ___ because ___ ___ is ____

allosteric site, not the active site bind, but enzyme function is impaired increasing [S] because the inhibitor affects enzyme activity, not binding decreases because the enzyme is rendered less effective stays the same because substrate binding is unaffected

Inhibitors: Uncompetitive: inhibitor binds only to the ___-___ ___ (__), not the ___ ____ prevents the enzyme from ___ ____, ___ the ____ inside only occurs when the ___ is already ___ both Vmax and Km ____ because the inhibitor locks the enzyme in the ___ ___ cannot be overcome by adding ___ ___ (since the inhibitor affects only the __ ___)

enzyme-substrate complex (ES), not the free enzyme releasing product, trapping the substrate inside substrate is already bound decrease because the inhibitor locks the enzyme in the ES state more substrate (since the inhibitor affects only the ES complex)

Lineweaver-Burk Plots: the lineweaver-burk plot is used to analyze enzyme kinetics by transforming the michaelis-menten equation into a ___ ____ Vmax (maximum ___ ___) Km (Michaelis constant, indicating ___ ___ for the ___)

linear form reaction velocity enzyme affinity for the substrate

Lineweaver-Burk Plots Michaelis-menten equation: describes how enzyme velocity (V0) depends on the ____ ___ [__] Vmax is the ___ ___ when all ___ ___ are ____ Km is the ___ ___ at ____, indicating how ___ an enzyme ___ to its ___

substrate concentration [S] maximum velocity when all enzyme sites are occupied substrate concentration at Vmax/2, indicating how strongly an enzyme binds to its substrate

Lineweaver-Burk Plots linear equation follows y=mx+b where: x-intercept = ___ y = ___ b = ____ m = ____ (slope) x = ____

x-intercept = -1/Km y = 1/V0 b = 1/Vmax m = Km / Vmax (slope) x = 1/[S]

inhibitor summary, lineweaver burk plots: competitive inhibition: Km ____ Vmax ____ slope ____ y-intercept ____

increased unaffected change same

inhibitor summary, lineweaver burk plots: uncompetitive inhibition: Km ____ Vmax ____ slope ____ y-intercept ____

reduced reduced same change

inhibitor summary, lineweaver burk plots: noncompetitive inhibition: Km ____ Vmax ____ slope ____ y-intercept ____

unaffected reduced change change

inhibitor summary, lineweaver burk plots: vmax is the maximum ___ of ____ when the enzyme is ___ with ___ Km is the ___ of ___ which permits the ___ to achieve _____

rate of reaction when the enzyme is saturated with substrate concentration of substrate which permits the enzyme to achieve Vmax/2

enzyme efficiency: Km: indicates ___ ___ for the ___ Kcat (__ ___): the maximum number of ___ ___ converted to ____ per ___ per ___ Kcat / Km (___ __): measures how ___ and enzyme converts a ___ into ___

enzyme affinity for the substrate turnover number: the maximum number of substrate molecules converted to product per enzyme per second catalytic efficiency: measures how efficiently an enzyme converts a substrate into product

enzyme efficiency: turnover number = "the maximum number of ____ ___ converted to ____ by a single ___ per unit ____, when E is ___ ___ with ____" = ___ = ___/___

substrate molecules converted to product by a single enzyme per unit time, when E is fully saturated with S" = kcat = Vmax / [ET]

enzyme efficiency: michaelis constant: Km = ___ at ___ (has units of ___) 1/Km provides the enzymes ___ for ____

Km = [S] at Vmax/2 (has units of M) afinity for substrate

enzyme efficiency: enzyme efficiency: ___/____ --> increases with ____ kcat or ___ Km also known as the "__ __ __", where high value = high ___ for S when [S] is ___

kcat/Km --> increases with larger kcat or smaller Km enzyme specificity constant, where high value = high specificity for S when [S] is low

enzyme efficiency: some enzymes catalyze reactions at "___-___ ___", turning over the substrate ___ ___ ____ it ___ some enzymes are so efficient that their reaction rate is limited only by how fast __ ___ can ___ into the ___ ___ this is known as the ___-____ ____, where kcat / Km approaches ____

diffusion-controlled limit, turning over the substrate as soon as it binds substrate molecules can diffuse into the active site diffusion-controlled limit, where kcat/Km approaches 10^9 M^-1s^-1

competitive inhibitors: increasing [I] ____ apparent Km because I ____ S from ___ ___ at sufficiently high [S], S ____ I and ___ is attained competitive inhibitors occupy the ___ ___, preventing ___ from ____

increases apparent Km because I blocks S from active site outcompetes I and Vmax is attained active site, preventing substrate from binding

uncompetitive inhibitors: increasing [I] ___ apparent Km by ___ S ____ increasing [I] ____ apparent Vmax by ____ [ES] uncompetitive inhibitors bind ____ to the ____ state, changing ____ and ____

decreases apparent Km by stabilizing S binding decreases apparent Vmax by depleting [ES] only to the ES state, changing Km and Vmax

mixed and noncompetitive inhibitors: mixed inhibitor can bind to both ___ and ____ mixed inhibitor ____ Vmax, just like ____ noncompetitive: ____ = ____, so Km does ___ ___, but Vmax ____ noncompetitive inhibitors bind at a ____ (___) site, only affect ___ ____ after noncompetitive inhibitor leaves, enzyme behaves ____, so value of ____ and ____ is unchanged, but Vmax ___

E and ES decreases Vmax, just like uncompetitive Ki = k'i, so Km does not change, but Vmax decreases second (allosteric) site, only affect product formation normally, so value of Km and 1/Km is unchanged, but Vmax decreases

inhibitor summary: competitive and uncompetitive inhibition are special cases of ____ ____ where Ki and Ki' are ____ (no ____) fitting lineweakver-burk plots is used to identify the mechanism of inhibition and determine ___ and/or ___

mixed inhibition, where Ki and Ki' are infinite (no binding) Ki and/or KI'

boronate protease inhibitors: boronates bind tightly to ___ ____ because they mimic the ____ ___ ____ the lineweaver-burk plot shows this to be competitive inhibition

ser proteases bc they mimic the oxyanion transition state

dual substrate reactions: dual substrate reactions can also be analyzed by ____-____ plots

lineweaver-burke plots

allosteric modulation: substrate modulation operates similarly to ____ ___ in hemoglobin modulators can be positive (___ activity) or negative (___ activity) modulators cause a ___ ____ that affects the ____ in the ___ ____

O2 binding in hemoglobin (increasing activity) or negative (decreasing activity) structural change that affects the site in the other subunit

allosteric modulation: allosteric modulation via substrate binding: some enzymes exhibit cooperative binding, meaning that binding of a substrate ____ the affinity of ____ substrate molecules this works similarly to O2 binding to ____, where O2 binding ____ hemoglobin's affinity for ____ O2 substrate binding induces a ____ ___, increasing enzyme activity

enhances the affinity of additional substrate molecules hemoglobin, where O2 binding increases hemoglobin's affinity for more O2 structural change, increasing enzyme activity

allosteric modulation: allosteric modulation via regulatory proteins or domains: some enzymes requires ____ ____ (___) to regulate their activity the R (____ domain/protein) interacts with ____ to control enzyme function positive modulator binds to ____ ____/___, inducing a ___ ___

external molecules (modulators) to regulate their activity (regulatory domain/protein) interacts with modulators to control enzyme function regulatory domain/protein, inducing a conformational change

allosteric effects on catalysis: allosteric modulation effects can be seen in plots of ____ ____ vs. ___ modulation can affect ___ (__), ___-____ (___), or ____ effects on "Vmax only" are rare, but can be seen in "___" ___ ___

reaction rate vs. [S] activity (Vmax), substrate-binding (Km), or both "pure" noncompetitive inhibition

allosteric effects on catalysis: positive modulators ____ enzyme function (either by ____ K0.5, or ___ Vmax) negative modulators ___ enzyme efficiency (by ____ K0.5, or ___ Vmax) modulation of K0.5 resembles ____ inhibition, while modulation of Vmax resembles ____ inhibition pure Vmax - only changes are rare and mostly seen in ___ ___-____ inhibition

enhance enzyme function (either by lowering K0.5, or increasing Vmax) reduce enzyme efficiency (by increasing K0.5, or decreasing Vmax) competitive inhibition, while modulation of Vmax resembles non-competitive inhibition pure non-competitive inhibition

monitoring an enzymatic reaction: chromatography: reactants and products can be separated by ____ and quantified using a ____ detector chromatograms run at ____ ____ can be used to determine the ___ of a ____ (__)

chromatography and quantified using a UV/Vis detector varying times can be used to determine the rate of a reaction (k)

testing an enzyme mechanism: protease: crystal structures may suggest certain ___ ____ carry out a reaction ___ of those ___ ___ can be used to testy that hypothesis by comparing ___ ___ of wild type and mutant enzymes if ser 195 acts as a ____, Ala mutant should be ____

amino acids carry out a reaction mutations of those amino acids can be used to testy that hypothesis by comparing reaction rates of wild type and mutant enzymes nucleophile, Ala mutant should be inactive