BCEM 393 - Enzymes [UNFINISHED]

What are catalysts?

Chemicals that enhance the rate of reactions without being permanently altered themselves

True or False: the role of enzymes is to make biochemically required reactions take place at a rate compatible with life.

True

Fill in the blanks: because of their specificity, enzymes bring _______ together at a particular site on the enzyme, called the ________, where they are oriented to facilitate the making and breaking of chemical bonds.

Substrates, active site

Fill in the blanks: some enzymes are information sensors as well as catalysts. In addition to active sites, ________ enzymes have distinct regulatory sites that bind to environmental signals. This binding modifies the activity of the _________.

Allosteric, active site

True or False: the binding of environmental signals to regulatory sites on allosteric enzymes modifies the activity of the active site.

True

Why must enzymes display specificity?

Enzyme specificity means a particular reactant should always yield a particular product. This minimizes side reactions leading to the formation of useless or hazardous by-products

True or False: most reactions in biological systems are able to take place at perceptible rates in the absence of enzymes, but enzymes help them proceed even faster.

False; most reactions in biological systems do not take place at perceptible rates in the absence of enzymes

Fill in the blanks: enzymes are highly specific both in the _____________ and in their choice of ________.

Reactions that they catalyze, substrates

Fill in the blanks: the specificity of an enzyme is due to the precise interaction of the ________ with the _______. This precision is a result of the ___________ of the enzyme protein.

Substrate, enzyme, intricate three-dimensional structure

What is absolute specificity?

Enzymes that catalyze only one reaction with a specific substrate

What is group specificity?

Enzymes that catalyze a few different reactions with molecules that have a specific functional group

Fill in the blanks: enzymes have varying degrees of specificity for their substrate. Some have absolute specificity, meaning the enzymes catalyze ________ with ________. Some have group specificity, meaning the enzymes catalyze _________ with _________.

Only one reaction, a specific substrate, a few different reactions, molecules that have a specific functional group

Fill in the blanks: enzymes have varying degrees of specificity. For example, papain is __________. Trypsin, a digestive enzyme, is _________. Thrombin, an enzyme that participates in blood clotting, is ________.

Very undiscriminating (it will cleave any peptide bond with little regard to the identity of the adjacent side chains), quite specific (catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues), even more specific (catalyzes the hydrolysis of Arg-Gly bonds in particular peptide sequences only)

True or False: enzymes speed up the rate of chemical reactions, but the properties of the reaction - whether it can take place at all - depends on free-energy differences.

True

What is Gibbs free energy?

The energy that is available to do work

Fill in the blanks: ΔG is the ________ difference between ________ and __________.

Free energy, products, reactants

Fill in the blank: ΔG tells us _____________.

Whether a reaction will happen under a set of conditions

Fill in the blank: at equilibrium, ΔG equals ____.

Zero

True or False: a spontaneous reaction has a +ΔG.

False; a spontaneous reaction has a -ΔG (exergonic)

Fill in the blanks: an __________ reaction requires the input of energy, while an __________ reaction releases energy.

Endergonic, exergonic

What is the formula for calculating Gibbs free energy change (ΔG)?

ΔG = ΔG°’ + RTln ( [C] [D] / [A] [B] )

where:

• R = gas constant

• T = temperature

• A + B ←→ C + D

What is the formula for calculating standard free energy change (ΔG°’)?

ΔG°’ = -RTln ( [C] [D] / [A] [B] )

where:

• R = gas constant

• T = temperature

• A + B ←→ C + D

Fill in the blanks: ___________ in the context of thermodynamics means that the reaction will take place without the input of energy, and that the reaction releases energy. These reactions are ___________.

Spontaneously, exergonic

True or False: for a reaction to be energetically favourable (spontaneous), ΔG must be negative.

True

Fill in the blanks: to understand how enzymes operate, two thermodynamic properties of the reaction must be considered: (1) the ___________ between the products and the reactants and (2) the ___________ required to initiate the conversion of reactants into products. The former determines whether the reaction will _____________ and the latter determines the _______________.

Free energy difference, free energy, take place spontaneously, rate of the reaction

True or False: the free energy difference between the products and the reactants determines the rate of the reaction.

False; the free energy difference between the products and the reactants determines whether the reaction will take place spontaneously. The free energy required to initiate the conversion of reactants into products determines the rate of the reaction

Fill in the blanks: the free energy change provides information about the __________ of a reaction but not the __________ of a reaction.

Spontaneity, rate

Fill in the blanks: a reaction cannot take place spontaneously if the free energy difference is ___________. An input of free energy is required to drive such a reaction. These reactions are called ____________.

Positive, endergonic

True or False: spontaneous reactions are instantaneous.

False; spontaneous reactions are NOT instantaneous

True or False: enzymes shift the equilibrium of a reaction.

False; enzymes do not shift the equilibrium of a reaction. The same equilibrium point is reached but faster

Fill in the blanks: in a system at equilibrium, there is __________ in the concentrations of the products and reactants and the free energy difference is ___________.

No net change, zero

Fill in the blanks: the free energy difference of a reaction depends only on the free energy of the __________ minus the free energy of the __________.

Products (final state), reactants (initial state)

True or False: the free energy difference provides no information about the rate of a reaction.

True

True or False: whether the ΔG for a reaction is larger, smaller, or the same as ΔG°’ depends on the concentrations of the reactants and products. Reactions that are not spontaneous, on the basis of ΔG°’, can be made spontaneous by adjusting the concentrations of reactants and products.

True

Fill in the blanks: enzymes accelerate reactions by decreasing the __________ to overcome the __________. Enzymes lower the activation energy by stabilizing the ___________.

Activation energy, activation energy barrier, transition state

Fill in the blanks: free energy is released when many __________ occur between the enzyme and the _________. This free energy is called _________, and its peak occurs during the __________.

Weak interactions, substrate, binding energy, transition state

True or False: maximal binding energy occurs during the transition state.

True

True or False: residues forming the active site come from different positions in the sequence.

True

True or False: the active site makes up a relatively large volume of the total protein.

False; the active site makes up a small volume of the total protein

Fill in the blanks: the active site of enzymes forms many __________ with substrates. Specificity of substrate binding depends on the __________ of ________ in the active site.

Weak interactions, precise arrangement, atoms

What are cofactors?

Non-protein compounds that bind to proteins and are needed for biological activity. Includes coenzymes (vitamin derived organic molecules) and metals

Fill in the blanks: an enzyme without its cofactor is referred to as an _________. The complete, catalytically active enzyme is called a __________.

Apoenzyme, holoenzyme

Fill in the blanks: cofactors can be subdivided into two groups; small organic molecules derived from vitamins, called ________, and _________.

Coenzymes, metals

Fill in the blanks: some very important coenzyme cofactors include ________ (decarboxylation), _________ (redox reaction), _________ (redox reaction), _________ (2C unit carrier), and __________ (CO₂ carrier).

Thiamine pyrophosphate (TPP), flavin adenine nucleotide (FAD), nicotinamide adenine dinucleotide (NAD⁺), coenzyme a (CoA), biotin

Fill in the blanks: some very important metal cofactors include ____, _____, _____, _____, ______, _____, and _____.

Zn²⁺, Mg²⁺, Ni²⁺, Mo, Se, Mn^2+←→3+, K⁺

Fill in the blanks: tightly bound coenzymes are called _________. Loosely associated coenzymes are more like __________ because, like substrates and products, they bind to the enzyme and are released from it.

Prosthetic groups, cosubstrates

What is a prosthetic group?

A cofactor that is very tightly bound to the enzyme active site

What is a cosubstrate?

A cofactor that binds and releases with each reaction

Fill in the blanks: coenzymes are distinct from normal substrates not only because they are often derived from ________ but also because they are _____________.

Vitamins, used by a variety of enzymes

True or False: different enzymes that use the same coenzyme usually carry out similar chemical transformations.

True

Fill in the blanks: there are two models of enzyme-substrate binding. One is the __________ model, in which the active site of the unbound enzyme is complementary in shape to the substrate. The other is the __________ model, in which the active site forms a shape complementary to the substrate only after the substrate has been bound.

Lock and key, induced-fit

Fill in the blanks: the study of the rates of chemical reactions is called ___________, and the study of the rates of enzyme-catalyzed reactions is called ___________.

Kinetics, enzyme kinetics

What is a first order reaction in enzyme kinetics?

Reactions in which the velocity is directly proportional to the reactant concentration are first-order reactions.

The velocity (V) of the reaction is the quantity of reactant A that disappears in a specified unit of time (t). It is equal to the velocity of the appearance of product (P), or the quantity of P that appears in a specified unit of time:

V = -d [A] / dt = d [P] / dt

Where d is the decrease in substrate concentration or the increase in product concentration.

The velocity of the reaction is directly related to the concentration of A by a proportionality constant k, called the rate constant:

V = k [A]

What is a second order reaction in enzyme kinetics?

Reactions with two reactants are second-order reactions. For example:

2 A → P or A + B → P

The corresponding rate equations often take the form:

V = k [A]² and V = k [A] [B]

Fill in the blanks: sometimes second-order reactions can appear to be first-order reactions. For instance, in V = k [A] [B] if the concentration of B greatly exceeds that of A and if A is present at low concentrations, the reaction rate will be _________________. These reactions are called ___________.

First order with respect to A and will not appear to depend on the concentration of B, pseudo-first-order reactions

Fill in the blank: the initial velocity of catalysis is defined as the _______________.

Number of moles of product formed per second shortly after the reaction has begun

Fill in the blank: the initial velocity of catalysis varies with the _________ when enzyme concentration is constant.

Substrate concentration

What is Michaelis-Menten kinetics?

Establishes a mathematical relation between V₀, V_max, and K_M.

E + S ←_k-1 ^k1→ ES ←_k-2 ^k2→ E + P

Where an enzyme (E) catalyzes the conversion of substrate (S) into product (P), and k1 is the rate constant for the formation of the enzyme-substrate (ES) complex, k2 is the rate constant for the formation of P, and k-1 and k-2 are the constants for the respective reverse reactions.

The amount of product formed is determined as a function of time for a series of substrate concentrations. The amount of product formed increases with time, but eventually a time is reached when there is no net change in the concentration of S or P. The enzyme is still actively converting substrate into product and vice versa, but the reaction equilibrium has been attained:

V₀ = V_max ([S] / [S] + K_M)

Where the Michaelis-Menten constant, K_M, is:

K_M = (k_-1 + k₂) / k₁

Fill in the blanks: Michaelis and Menten proposed a simple model to account for enzyme kinetic characteristics. The critical feature in their treatment is that a __________ is a necessary ________ in catalysis.

Specific ES complex, intermediate

Fill in the blanks: in the Michaelis-Menten model, the _________ state is the initial mixing of E + S, while [ES] builds up, and the _________ state is when [ES] remains approximately constant. ______________ describes measurements of V₀ while [ES] is relatively stable.

Pre-Steady, Steady, Steady-State Kinetics

Fill in the blanks: ____, a compilation of rate constants called the _____________, is unique to each enzyme and is independent of enzyme concentration. It describes the properties of the enzyme-substrate interaction and thus varies for enzymes that can use different substrates.

K_M, Michaelis-Menten constant

Fill in the blanks: in Michaelis-Menten kinetics, the maximal velocity possible, _____, can be attained only when all of the enzyme, ____________, is bound to substrate (S), so _______=________.

V_max, total enzyme (E_T), V_max = k₂ [E]_T

True or False: in Michaelis-Menten kinetics, V_max is directly dependent on enzyme concentration.

True

Fill in the blanks: in Michaelis-Menten kinetics, at very low substrate concentrations, when [S] is much less than the value of K_M, the velocity is directly proportional to the substrate concentration; that is, ___________.

V₀ = (V_max / K_M) [S]

Fill in the blanks: in Michaelis-Menten kinetics, at high substrate concentrations, when [S] is much greater than K_M, ___________; that is, the _________ is __________, independent of ___________.

V₀ = V_max, velocity, maximal, substrate concentration

Fill in the blanks: when an enzyme is operating at Vmax, all of the available enzyme is ___________; the addition of more _________ will not affect the velocity of the reaction. Under these conditions, the enzyme is said to be ___________.

Bound to substrate, substrate, saturated

Fill in the blanks: in Michaelis-Menten kinetics, when [S] = K_M, V₀ = __________, so V₀ = __________, so K_M is equal to the [S] at which the reaction rate is _________ its maximal value.

V_max ([S] / [S] + [S]), V_max / 2, half

Fill in the blanks: K_M depends on __________, __________, __________, and __________.

Substrate identity, pH, temperature, ionic strength

True or False: K_M is the concentration of substrate at which all of the enzyme molecules are bound to substrate.

False; K_M is the concentration of substrate at which half of the enzyme molecules are bound to substrate

True or False: the cellular concentration of a particular substrate is often close to the K_M value.

True

What is the Lineweaver-Burk equation?

A simple, but often slightly inaccurate, method for determining KM and Vmax before the availability of computers.

1 / V₀ = K_M / V_max x 1 / S + 1 / V_max

Fill in the blanks: for most enzymes, K_M lies between _____ and _____.

10^-1, 10^-7

Fill in the blanks: in the Lineweaver-Burk equation, 1 / V₀ = (K_M / V_max) x (1 / [S]) + (1 / V_max), 1 / V₀ corresponds to __ (______), K_M / V_max corresponds to __ (_____), 1 / [S] corresponds to __ (_____), and 1 / V₀ corresponds to __ (_____) in the line equation y = mx + b.

y, Y-axis, m, slope of the line, x, X-axis, b, where the line crosses the Y-axis

Estimate the KM and Vmax from a Lineweaver-Burk plot with the equation Y = 0.6449x + 0.1934.

The linear Michaelis-Menten equation is:

1/V₀ = (K_M / V_max) x (1 / [S]) + (1 / V_max)

Which aligns with y = mx + b, so,

m = 0.6449 = K_M / V_max

b = 0.1934 = 1 / V_max

V_max = 1 / 0.1934 = 5.17

K_M = 0.6449 x V_max = 0.6449 × 5.17 = 3.34

Multiple Choice: the Michaelis constant (K_M) depends on _________ (select all that apply)

a) Enzyme concentration

b) Type of substrate

c) [H⁺] in the reaction buffer

d) Type of enzyme

e) Temperature

b), c), d), e)

True or False: for many enzymes, experimental evidence suggests that the K_M value provides an approximation of substrate concentration in vivo, suggesting that most enzymes evolved to have significant activity at the substrate concentration that is normally available for that enzyme.

True

Define: turnover number (k_cat).

The number of substrate molecules that an enzyme can convert into product per unit time when the enzyme is fully saturated with substrate

Fill in the blanks: the turnover number is equal to __________, which is also called ____, for ___________.

The rate constant (k₂), k_cat, the rate of catalysis

Fill in the blanks: if the total concentration of active sites ([E]_T) is known, then V_max = k₂ [E]_T, and thus, k₂ (aka k_cat) = __________.

V_max / [E]_T

Fill in the blanks: when the substrate concentration is much lower than K_M, the enzymatic rate is ___________ than k_cat (k₂) because _______________. Under these conditions, the equation _____________ can be derived. Here, the rate constant k_cat / K_M, called the __________, is a measure of catalytic efficiency because it takes into account both the rate of catalysis with a particular substrate and the nature of the enzyme-substrate interaction.

Much less, most of the active sites are unoccupied, V₀ = (k_cat / K_M) [S] [E]_T, specificity constant

Fill in the blanks: when [S] « K_M, then V₀ = (k_cat / K_M) [S] [E]_T. Under these conditions, k_cat / K_M is the rate constant for ____ formation, and is called the __________ because it is a measure of _________ because it takes into account rate of catalysis (k_cat) and the nature of the enzyme-substrate interaction (K_M).

ES, specificity constant, catalytic efficiency

Fill in the blank: by using _________, an enzymes preference for different substrates can be compared.

k_cat / K_M values

Fill in the blanks: the ultimate limit on the value of k_cat / K_M, which shows enzyme efficiency, is set by ____, the ____________. _________ limits the value of ____, so it cannot be higher than 10⁸ to 10⁹ s^-1 M^-1, hence, this is the upper limit on catalytic efficiency (k_cat / K_M).

k₁, rate of formation of the enzyme-substrate (ES) complex, diffusion, k₁

Fill in the blanks: enzymes that have k_cat / K_M ratios at the upper limits (10⁸ to 10⁹ s^-1 M^-1) have attained ___________. Every encounter between substrate and enzyme is productive. Their catalytic velocity is restricted only by _______________.

Kinetic perfection, the rate at which they encounter substrate in the solution

Fill in the blanks: enzyme activity _________ with temperature until the enzyme ____________.

Increases, denatures

True or False: the optimal pH for enzyme activity is at around 2.

False; the optimal pH for enzyme activity varies and reflects the environment (e.g., ~1.5 for pepsin (in stomach), ~8 for chymotrypsin (in proximal small intestine))

Fill in the blanks: enzymes that conform to simple Michaelis-Menten kinetics are called Michaelis-Menten enzymes These enzymes are not regulated in the cell, and their activity is governed simply by mass action; if __________ is __________, they ___________. Most enzymes in the cell are Michaelis-Menten enzymes.

Substrate, present, catalyze

Define: allosteric enzymes

Enzymes that regulate the flux of biochemicals through metabolic pathways. They catalyze committed steps of biochemical pathways (e.g., A → e1 (allosteric enzyme) → B → e2 → C → e3 → D, where B is the committed step; after this reaction, B is committed to conversion into D)

Fill in the blanks: allosteric enzymes have a __________ structure and __________ active site(s). They can bind both __________ molecules and __________ molecules at ____________.

Quaternary, multiple, inhibitory, stimulatory, regulatory sites

Fill in the blanks: inhibitors bind to ___________ on allosteric enzymes, which ____________ enzyme activity, signaling that there is _____________ and turns the pathway _____.

Regulatory sites, decreases, enough product, off

True or False: allosteric enzymes always catalyze the committed step of metabolic pathways.

True

Fill in the blanks: a metabolite produced early in the metabolic pathway binds to a ____________ on an allosteric enzyme down stream in the pathway, signaling a(n) ___________ in substrate, which ___________ enzyme activity.

Regulatory site, increase, increases

True or False: allosteric enzymes follow Michaelis-Menten kinetics.

False; allosteric enzymes display sigmoidal kinetics, and do not follow Michaelis-Menten kinetics

Fill in the blanks: allosteric enzymes display ____________ kinetics. There are two models for explaining _____________ kinetics; ___________ and ___________.

Sigmoidal, sigmoidal, concerted, sequential

Fill in the blanks: in the concerted sigmoidal kinetics model for allosteric enzymes, enzymes have ____________ on different polypeptide chains, and a __________ form that catalyzes reactions and a ___________ form that is less active but more stable and more common, which are in equilibrium. The allosteric constant = _______ = L₀, and is typically in the hundreds.

Multiple active sites, relaxed (R), tense (T), T / R

Fill in the blank: in the concerted model for allosteric enzyme kinetics, there is a symmetry rule, which means _______________.

All active sites must be in the same state

Fill in the blanks: in the concerted model for allosteric enzyme kinetics, the substrate binds more readily to the ________ form than the __________ form.

Relaxed (R), tense (T)

Fill in the blanks: in the concerted model for allosteric enzymes, when [S] is low, most enzyme is in the __________ form. As [S] increases, it binds an _____________, trapping all other ___________ in ________ form. As more ____________ are in ________ form , it is easier for substrate to bind.

Tense (T), active site on R, active sites, relaxed (R), active sites, relaxed (R)

Fill in the blanks: in the concerted model for allosteric enzyme kinetics, the binding of substrate shifts the T ←→ R towards the _________ form. This behaviour is called _____________.

Relaxed (R), cooperativity

Fill in the blanks: allosteric enzymes are ________ sensitive to changes in [S] near K_M than Michaelis-Menten enzymes with the same V_max. This is the _______________.

More, threshold effect