Biochemistry Exam #3

What are enzymes?

biological catalysts

Draw the graph to compare energy & reaction co-ordinates for non-catalyzed & catalyzed reactions.

Explain the differences in the two types of energy between non-catalyzed & catalyzed reactions.

enzymes lower the activation free energy, but does not change the overall free energy

List 3 functions of enzymes that allow for lower activation energy & thus an easier reaction.

• stabilizing the transition state & lowering the activation barrier

• providing an alternative path for product formation which could include the formation of a stable, reaction intermediate

• orienting the substrate so that it is easier for the reaction to occur (collision of substrate molecules)

What is the active site of an enzyme?

where the substrate binds & reactive groups align correctly for reaction to occur & product to form

What holds substrates in the active sites of enzymes?

multiple, weak, non-covalent interactions

What is the induced-fit mechanism?

the structure of an enzyme changes in order to accommodate substrate binding

What is a co-factor?

small molecules aiding catalytic reactions at the active site

List two examples of co-factors.

• inorganic ions (Mg²⁺, Fe²⁺, Cu²⁺)

• co-enzymes

What are co-enzymes?

co-factors with an organic component

List an example of a co-enzyme.

vitamin derived species (NAD+, FAD+)

What are prosthetic groups?

co-enzymes that are permanently associated with enzymes

List two examples of prosthetic groups.

• NADP

• heme

Differentiate between apoenzymes & holoenzymes.

• apoenzyme: enzymes lacking their co-factors; inactive

• holoenzyme: enzymes containing co-factors; active

How are enzyme classes defined?

by function of the enzyme

List six examples of enzyme classes.

• oxidoreductase

• transferase

• hydrolase

• lyase

• isomerase

• ligase

List the function of each class of enzyme.

• oxidoreductase: catalyze redox reactions by transferring hydrogen or oxygen atoms

• transferase: transfer of functional groups

• hydrolase: formation of two products by hydrolyzing a substrate

• lyase: cleavage of bonds via means other than hydrolysis or oxidation

• isomerase: intramolecular rearrangements via transfer of groups within molecules

• ligase: ligation (forming of) bonds using ATP cleavage

What is enzyme kinetics?

the quantitive study of the rate of chemical reactions catalyzed by enzymes

Describe how the relevant parameters are related in regard to enzyme kinetics.

reaction rates are related to free energy through kinetic parameters

What is the purpose of the Michaelis-Menten equation?

it calculates the rate of reaction in terms of maximum rate of reaction (V_max) and Michaeli’s constant, Km

Write the enzyme-substrate reaction that the Michaelis-Menten equation derives from.

What does the reaction rate (v) vary with?

concentration of the substrate, [S]

What assumption is made to derive the Michaelis-Menten equation?

steady state

What is the steady state assumption?

the concentration of enzyme-substrate complex remains constant over the duration of the reaction

Express the steady state mathematically.

rate of formation of ES = rate of breakdown of ES

What is the equation for the rate of formation of ES?

rate of formation = k₁ [E] [S]

What is the equation for the rate of breakdown of ES?

rate of breakdown = k₂ [ES]+ k₃ [ES]

What is the simplified equation for the rate of breakdown of ES?

rate of breakdown = (k₂ + k₃) [ES]

At steady state, what is the relationship between equations of rate of formation of [ES] & rate of breakdown of [ES]?

k₁ [E] [S] = (k₂ + k₃) [ES]

What is the next step for deriving the Michaelis constant?

divide by (k₂+ k₃) → k₁ [E] [S] / k₂ + k₃ = [ES] and then divide by k₁ to get → [E] [S] / k₂ + k₃/k₁ = [ES]

What is Michaelis constant, k_m?

k₂ + k₃ / k₁

Now substitute k_m in.

[E] [S] / k_{m =} [ES]

Rearrange k_m again.

[E] [S] = k_m [ES]

What is the Michaelis constant determined by?

the reaction rates of the formation & breakdown of the enzyme-substrate complex

Draw a graph describing the relationship between rate of reaction (v), substrate concentration [S], and maximum rate of reaction, v_max

Describe what is happening at the maximum rate of reaction.

when substrate concentration becomes high, all available enzymes become saturated with substrate and make max. product, P

What is the equation for v_max?

v_max= k₃ [E_t]

What is [E_t]?

total enzyme concentration, ( [E] + [ES]

What is the Michaelis-Menten equation?

v₀ = v_max [S] / k_m + [S]

What is v₀?

initial reaction rate

Derive the equation for k_m when v₀ is equal to v_max/2

Define the enzyme-substrate reaction using k_-1 notation.

What is the general equation for k_m, Michaelis constant?

k_-1 + k₂ / k₁

What is the equation for k_m in the circumstance that k₂ is much smaller than k_-1 ?

k_m = k_-1/k₁

What is this defined as?

the disassociation constant, k_D

In this situation, what is k_m a measure of?

an approximate measure of affinity of enzyme for substrate

What does a lower k_m mean?

the enzyme is better at working at low substrate concentrations, hence it has a higher affinity for the substrate

What does a higher k_m mean?

the enzyme is better at working at high substrate concentrations, hence it has a lower affinity for the substrate

What is k_m related to?

catalytic efficiency

What is catalytic efficiency?

the enzyme’s efficiency in catalyzing the reaction

What is k_cat?

how many substrate molecules an enzyme can turnover into product in one second

What is the equation for k_cat including units?

k_cat = v_max (moles/s) /E_t (moles)

What is the unit for k_cat?

1/s or s^-1

What measure determines catalytic efficiency?

the specificity constant

What is the equation for the specificity constant?

k_cat/k_m

What is the relationship between different enzymes & catalytic efficiency?

each enzyme has a different catalytic efficiency

For greater catalytic efficiency, is a higher or lower k_cat better? What about k_m?

higher k_cat, lower k_m

What do you need to do to determine which of two different enzymes for the same reaction is more efficient?

compare the catalytic efficiency of both using k_cat/k_m; the higher value is the more efficient enzyme

Describe how enzymes perform in regard to temperature & pH conditions.

enzymes perform at optimal pH & temperature

Explain the function of pepsin & its optimal pH condition.

function: breaks down dietary proteins

optimal pH condition: high acidic environments ~ 1.6 pH

Explain the function of glucose-6-phosphatase, where it is present, and its optimal pH conditions.

function: breaks down glucose-6-phosphate

location: endoplasmic reticulum

optimal pH condition: ~7.8 pH

Explain the function of arginase, where it is present, and its optimal pH conditions.

function: involved in urea cycle in the kidney

location: mitochondria

optimal pH conditions: ~9.7 pH

Explain generally how pH can affect the functionality of an enzyme.

pH can change the charges on an amino acid side change due to acceptance or donation of H+, which can affect the structure, binding & catalytic reactions of that enzyme, causing the enzyme to be less active

Explain the difference in optimal pH conditions between E. coli DNA polymerase & tag DNA polymerase.

• E.coli DNA polymerase optimal temperature: 37°C

• tag DNA polymerase optimal temperature: 80°C

What is tag DNA polymerase used in? What is it derived from?

• PCR

• hot-spring bacteria

Explain generally how temperature can affect the functionality of an enzyme.

usually, increasing temperature increases molecular movement, allowing more frequent, productive collisions, but when temperature becomes too high, the enzyme can denature, losing the shape required for catalysis

What is the function of the Lineweaven-Burke plot?

it converts the Michaelis-Menten equation into a linear equation

Draw the Lineweaven-Burke plot using the Michaelis-Menten equation.

Where is k_m on this graph?

at the halfway point to v_max.

Why is it beneficial to invert this graph into linear form?

to more accurately determine the values of v_max & k_m.

Derive the inverted Michaelis-Menten equation.

Now align each part of the equation with the linear form, y=mx+c.

y = 1/v_o

m = k_m/v_max

x = 1/[S]

c = 1/v_max

What is the y-intercept?

What is the x-intercept?

Graph the Lineweaven-Burke plot showing all values.

What are enzyme inhibitors?

molecules that decrease or inhibit enzymatic activity

List the two types of enzyme inhibitors.

• irreversible enzyme inhibitors

• reversible enzyme inhibitors

Describe the functionality of irreversible enzyme inhibitors.

forms a covalent bond or a very strong non-covalent interaction at the enzyme active site with catalytic groups which kills (permanently inactivates) the enzyme

What are catalytic groups?

chemical groups, often amino acid side chains, that are directly involved with catalysis of substrate

Explain the functionality of reversible enzyme inhibitors.

have non-covalent interactions w/ the enzyme

How many types of reversible enzyme inhibitors are there & what are they classified based on?

• 3

• their interactions w/ the enzyme & enzyme-substrate complex

List the 3 reversible enzyme inhibitors.

• competitive inhibitors

• uncompetitive inhibitors

• mixed inhibitors

Describe the functionality of competitive enzyme inhibitors.

competitive inhibitors inhibit the free enzyme by competing with the substrate for binding at the active site, preventing substrate binding

How can competitive enzyme inhibitors be overcome?

excess substrate addition

Describe the functionality of uncompetitive enzyme inhibitors.

uncompetitive enzyme inhibitors bind to the enzyme-substrate complex, preventing product from being formed

Describe the functionality of mixed enzyme inhibitors.

a mix of competitive & uncompetitve; can bind to both the enzyme itself to prevent substrate binding or the enzyme-substrate com

plex to prevent product formation

What is noncompetitive enzyme inhibition?

a type of mixed enzyme inhibition in which the inhibitor binds with equal affinity to both the free enzyme & the enzyme-substrate complex

Describe the active site of an enzyme (2).

sequestered microenvironment that provides the optimal orientation of the substrate w/ the functional groups that interact w/ the substrate to catalyze the reaction

What results from binding of the substrate & enzyme?

formation of the transition state

What is the transition state?

a high energy arrangement of atoms that occurs as reactants are turned into products

How many different types of catalysis mechanisms are there?

3

List the 3 types of catalysis mechanisms.

• acid-base catalysis

• covalent catalysis

• metal-ion catalysis

Describe the functionality of acid-base catalysis.

involves proton transfer from water or a functional group on the enzyme

What is frequently involved in acid-base catalysis & why?

amino acid, histidine (his) because its side chain has a pka of 7, meaning it can form either an acid or a base easily

What is an example of acid-base catalysis?

RNA cleavage by ribonuclease

Explain the functionality of covalent catalysis.

a nucleophile group on the enzyme attacks an electrophile on the substrate to form a covalent enzyme-substrate intermediate

What is an example of covalent catalysis? Briefly explain how this mechanism works.

G3P dehydrogenase, which is an enzyme that contains cysteine whose sulphydryl group forms a covalent bond w/ glyceraldehyde-3-phosphate

Explain the functionality of metal-ion catalysis

a metal ion is used to promote the proper orientation of the bound substrate & help in re-dox reaction

What is an example of a metal-ion that aids in enzyme catalysis?

Zn²⁺ in carbonic anhydrase

Explain chymotrypsin mechanism. Which catalysis mechanisms are involved?

• serine is converted via proton removal to form a highly reactive nucleophile that helps in the cleavage of amide bonds

• covalent catalysis & acid-base catalysis