MCB 354 Exam #2

What is the main source of energy for enzymes to lower activation energy?

Substrate-enzyme binding energy

What is true about [ES] and conc. of other intermediates at steady state?

Constant

What is true about [S] at the beginning of the reaction?

Constant

What is the full Michaelis-Menten reaction sequence?

E + S ⇌ ES ⇌ E + P

Which step in the Michaelis-Menten reaction sequence is the rate-determining (slow) step?

Second step (ES ⇌ E + P)

What concentration term is the full Michaelis-Menten reaction sequence’s overall rate proportional to?

[ES]

How can V_max be interpreted in terms of the enzyme?

V_max is achieved when all of the enzyme is present in the ES complex

How does increasing [S] past saturation have an effect on V_max?

It has no effect, V_max plateaus due to enzyme concentration

What is the Michaelis-Menten equation?

V₀ = (V_max[S])/(K_m + [S])

What can the Michaelis-Menten reaction sequence be simplified to, and why?

E + S ⇌ ES → E + P, k₂ » k_-2

What is V₀ equal to, based on the rate-determining step of the simplified Michaelis-Menten reaction sequence?

k₂[ES]

Why is total enzyme concentration used as a substitution rather than [ES] directly?

[ES] is very difficult to measure experimentally

What is true about the relationship between [S] and [E]_t, and what is its interpretation?

[S] » [E]_t, so the amount of substrate bound by enzyme is negligble with the total [S]

What are the rate equations for ES formation and breakdown, using [E] = [E]_t - [ES]?

Formation: k₁([E]_t - [ES])[S]

Breakdown: k_-1[ES] + k₂[ES

What does the steady-state assumption state qualitatively?

Rates of ES formation and breakdown are equal

Mathematically, what is the steady-state assumption, and what does solving it for [ES] yield?

Assumption: k₁([E]_t - [ES])[S] = k_-1[ES] + k₂[ES]

[ES] = ([E]_t[S])/([S] + (k_-1 + k₂)/k₁)

What is the Michaelis constant equal to, in terms of the elementary rate constants?

(k_-1 + k₂)/k₁

What is V_max equal to in terms of [E]_t?

k₂[E]_t

On a plot of [S] vs. V₀, what is the interpretation of K_m?

K_m = [S] at ½ V_max

What is true about K_m for a multi-substrate enzyme?

K_m can be different for different substrates of the same enzyme

When can K_m be simplified into K_d, the dissociation constant?

When k₂ is rate-limiting, i.e., k₂ « k_-1

What does K_d represent?

Affinity of the enzyme for the substrate

What does k_cat represent?

Turnover number, the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated

What does k_cat equal in the Michaelis-Menten equation?

V_max/[E]_t

What is the Michaelis-Menten equation re-written with k_cat?

V₀ = (k_cat[E]_t[S])/(K_m + [S])

What rate constant order is k_cat?

First order

What is the best method of comparing overall catalytic efficiencies of two enzymes?

Compare their ratios k_cat/K_m (the specificity constant), where a higher ratio → higher efficiency

When does the specificity constant equal the overall rate constant for E + S → E + P, and what is the Michaelis-Menten equation in that case?

[S] « K_m, V₀ = k_cat/K_m [E]_t[S]

What order rate constant is the specificity constant?

Second order

How is the Lineweaver-Burk equation derived?

Take the reciprocal of both sides of the Michaelis-Menten equation

What does the slope of the Lineweaver-Burk equation equate to?

K_m/V_max

What do the y- and x-intercepts of the Lineweaver-Burk equation equate to?

y-intercept: 1/V_max

x-intercept: -1/K_m

What is the “ping-pong” enzymatic mechanism?

An enzyme-catalyzed reaction where the first product is released before the second substrate binds, the enzyme is modified after the first product is released, and release of the second product reverts the enzyme to its first state

What are the rules for Cleland nomenclature?

Substrates: A, B, C, and D, in the order in which they bind to the enzyme

Products: P, Q, S, and T, in the order in which they dissociate

Num. substrates: one, two, three, or four substrates = uni, bi, ter, and quad, respectively

Enzyme: E, but if modified during the reaction, successive forms = F, G, and so on

What do intersecting lines on a Lineweaver-Burk plot represent?

Formation of a ternary complex

What do intersecting lines on a Lineweaver-Burk plot represent?

“Ping-pong” mechanism

What does a burst of product concentration detected mean?

The rate-determining step comes after that product is already formed

What are the four types of reversible enzyme inhibitors?

Competitive, uncompetitive, mixed, non-competitive

Where in the Michaelis-Menten reaction sequence does a competitive inhibitor bind?

Binds E in E + S

What is the Michaelis-Menten equation for a competitive inhibitor?

V₀ = (V_max[S])/(αK_m + [S]), α = 1 = [I]/K_I, K_I = ([E][I])/[EI]

What is the effect on K_m in the presence of a competitive inhibitor?

K_m is scaled by α

What is the effect on V_max in the presence of a competitive inhibitor?

No effect on V_max

How can the presence of a competitive inhibitor be visualized on a Lineweaver-Burk plot?

Lines intersecting at y-axis

Where in the Michaelis-Menten reaction sequence does an uncompetitive inhibitor bind?

Binds ES in the ES complex step

What is the Michaelis-Menten equation for an uncompetitive inhibitor?

V₀ = (V_max[S])/(K_m + α’[S]), α’ = 1 = [I]/K’_I, K’_I = ([ES][I])/[ESI]

What is the effect on K_m in the presence of an uncompetitive inhibitor?

Apparent K_m decreases, K_m/α’

What is the effect on V_max in the presence of an uncompetitive inhibitor?

V_max decreases, V₀ approaches V_max/α’ at high [S]

How can the presence of an uncompetitive inhibitor be visualized on a Lineweaver-Burk plot?

Parallel lines

Where in the Michaelis-Menten reaction sequence do mixed and noncompetitive inhibitors bind?

Binds to E in E + S and ES in ES complex step

What is the Michaelis-Menten equation for a mixed or noncompetitive inhibitor?

V₀ = (V_max[S])/(αK_m + α’[S])

What is the effect on K_m in the presence of a mixed or noncompetitive inhibitor?

Apparent K_m may increase or decrease depending on which form of the enzyme the inhibitor binds most strongly, no effect on K_m, αK_m/α’

What is the effect on V_max in the presence of a mixed or noncompetitive inhibitor?

V_max decreases, V_max/α’

What is the difference between mixed and noncompetitive inhibitors?

Noncompetitive inhibitors are special cases of mixed inhibitors where α = α’ and K_m is unaffected

What is an irreversible inhibitor?

An inhibitor that covalently binds or destroys an enzyme

What is a suicide inactivator?

A class of irreversible inhibitor that relies on chemical transformation to turn into the irreversible inhibitor

What is a transition-state analog?

Stable molecules that resemble transition states and very tightly bind the enzyme, even more tightly than the substrate

What is the difference between the acylation and deacylation phase of chymotrypsin?

Acylation: peptide bond is cleaved, ester linkage formed between peptide carbonyl carbon and enzyme

Deacylation: ester linkage is broken and nonacylated enzyme is regenerated

At what pH is chymotrypsin activated, and what are the protonation states of its catalytic residues?

His⁵⁷ is deprotonated and Ile¹⁶ is protonated

What are serine proteases?

Proteases with a Ser residue that acts as the nucleophile

How does acylated chymotrypsin act as a serine protease?

Ser¹⁹⁵ oxygen acts as a nucleophile

What is the catalytic triad in the example of chymotrypsin, and how are the three residues linked?

Ser¹⁹⁵, His⁵⁷, Asp¹⁰², linked by H-bonds

What type of enzyme mechanism is chymotrypsin?

Ping-pong

What reaction does hexokinase catalyze?

Glucose → glucose 6-phosphate

What enzyme mechanism does hexokinase demonstrate, and how?

Induced fit, binding energy of glucose and Mg • ATP causes a conformational change to the catalytically active form

What reaction does enolase catalyze?

Reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate

What enzyme mechanism dose enolase demonstrate, and how?

Metal ion catalysis, Mg²⁺ ions stabilize enolate intermediate formed by base reaction with Lys³⁴⁵, then causing removal of OH by Glu²¹¹ via an acid reaction

What activates and what inactivates aspartate transcarbamoylase?

ATP activates, CTP inactivates

How do allosterically regulated enzymes deviate from Michaelis-Menten?

Sigmoidal rather than hyperbolic curve

What effect do heterotropic allosteric regulators have on K_0.5?

Activators decrease, inhibitors increase

What effect do allosteric regulators have on V_max?

No effect

What conformational change does phosphorylation have on glycogen

phosphorylase?

Moves N-termini, and thus Ser¹⁴, to opposite side laterally

What proteins comprise the monomers of blood clots, and what are their zymogens called?

Fibrin, fibrinogen

What enzyme is responsible for the cleavage of fibrinogen into fibrin?

Thrombin (serine protease)

What enzyme is responsible for catalyzing cross-linking between fibrin?

Factor XIIIa (transglutaminase)

What is the difference between the intrinsic and extrinsic coagulation cascade?

Intrinsic: uses only components found in blood plasma

Extrinsic: also uses the protein tissue factor (TF), which is not found in blood

What is the difference between an aldose and a ketose?

Aldose: carbonyl group is at an end of the carbon chain

Ketose: carbonyl group is at any other position

These are based on Fisher projection

How many chiral centers do monosaccharides contain?

At least one

How can D- or L-stereoisomerism be determined from a Fischer projection?

D-stereoisomer: bottom chiral center -OH faces right

L-stereoisomer: bottom chiral center -OH faces left

What are epimers?

Two sugars that differ only in the configuration around one carbon atom

What is the anomeric carbon, and how can it be identified from a Fischer/Haworth projection?

Carbonyl carbon on Fischer projection, carbon next to ether O (but on the other side from the CH₂OH)

What is the difference between α and β anomers?

α: anomeric -OH is in the same direction as -CH₂OH

β: anomeric -OH is in the opposite direction as -CH₂OH

What is mutarotation?

Intracoversion between α and β anomers

What is the difference between furanoses and pyranoses?

Furanoses: 5-membered rings

Pyranoses: 6-membered rings

Describe the conversion from Fischer to Haworth projection.

Right on Fischer → down on Haworth

Left on Fischer → up on Haworth

CH₂OH up for D, down for L

Anomeric hydroxyl same direction as CH₂OH for α, opposite direction for β

What type of ring can undergo conformational changes without breaking covalent bonds, and is it energy-dependent?

Pyranoses, requires energy input

How can reducing sugars be identified from Haworth projection?

There is a free anomeric -OH

What common disaccharide is reducing?

Lactose

What common disaccharides are non-reducing?

Sucrose, trehalose

What polymers comprise starch, and what linkages do they exhibit?

Amylose (α1→4 between D-glucose) and amylopectin (α1→4 between D-glucose, α1→6 between chains)

What monomers comprise glycogen, and what linkages do they exhibit?

Glucose (α1→4 between D-glucose, α1→6 between chains)

What monomers comprise cellulose, and what linkages do they exhibit?

Glucose (β1→4)

What monomers comprise chitin, and what linkages do they exhibit?

N-acetylglucosamine (β1→4)

What is the chemical difference between chitin and cellulose, and what physical property does this lend chitin?

Chitin contains an acetylated amino group, making it more hydrophobic

What 3D structure does starch and glycogen adopt?

Helical with six residues/turn

What 3D structure does cellulose adopt?

Linear with each chair running antiparallel to the last

What monomers comprise peptidoglycan, and what linkages do they exhibit?

N-acetylglucosamine and N-acetylmuramic acid, (β1→4), crosslinked by short peptides

What monomers comprise the extracellular matrix?

N-acetylglucosamine or N-acetylgalactosamine and uronic acid

Describe the architecture of proteoglycans.

Sulfated glycosaminoglycan chains attached by a tetrasaccharide linker to Ser

How does antithrombin inhibit thrombin?

Antithrombin binds to thrombin in the presence of heparin sulfate due to both antithrombin/thrombin containing high numbers of Lys/Argm, allowing them to bind electrostatically to negatively-charged sulfates

What is the structure and function of fibronectin?

Contains domains to bind fibrin, heparin sulfate, collagen, and integrins; allows for signaling via integrins from cell interior to ECM