chapter 6

Question 1

Enzymes: What and Why?

Enzymes are
Most enzymes are
Regulatory enzymes help to
Enzymes function in
Many disease conditions can be related to

Answer 1

Enzymes are catalysts
Increase reaction rates without being used up

Most enzymes are globular proteins
-However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions

Enzymes function in aqueous systems (mild condition: 37°C and pH ≈7)

Regulatory enzymes help to coordinate metabolic pathways

Many disease conditions can be related to inappropriate levels of enzymatic activity

Question 2

Catalysis has high degree of
-MW:
-Some require additional chemical components for
and they are:

-Holoenzyme/apoenzyme or apoprotein

Answer 2

-Catalysis: high degree of specificity (3D conformation)
-MW: 12 kD to 1,000 kD
-Some require additional chemical components for activity – cofactor (one or more inorganic ions; Coenzyme)
-Coenzyme – complex organic or metalloorganic molecule acting as a functional group carrier
-Prosthetic group – a coenzyme or metal ion that is very tightly or covalently bound to the protein
-Holoenzyme-protein +cofactor in active form
apoenzyme or apoprotein- protein without cofactor

Question 3

Why biocatalysis over inorganic catalysts?

Answer 3

-Greater reaction specificity: avoids side products
-Milder reaction conditions: conducive to conditions in cells
-Higher reaction rates: in a biologically useful timeframe
-Capacity for regulation: control of biological pathways

Question 4

Enzyme Classification: 7 classes

Answer 4

1. Oxidoreductases- oxi reduces
2. Transferases-transfers
3. Hydrolases-hydrolysis
4. lyases-cleave
5. Isomerases-transfer groups in isometric forms
6. Ligases-glue
7. Translocases-translocate 1 molecule across a membrane

Question 5

How Enzymes Work: Enzyme-Substrate Complex

Answer 5

active site: the binding pocket on the enzyme where the enzyme-catalyzed reaction takes place
Substrate: the mol. bound in the active site and acted by the enzyme

Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin with bound substrate (PDB ID 7GCH) (Key aa: red).

Question 6

∆G‡:
∆G’°

Answer 6

∆G‡: the activation energies
∆G’°: the overall standard free-energy change (S  P)

Question 7

ES and EP:
Enzymes affect

Answer 7

ES and EP: transient complexes
Enzymes affect reaction rates, NOT equilibria, lower activation energy, small ∆G

Question 8

Reaction rates

Answer 8

S → P
V = k[S] (first order reaction)
k is a proportionality constant reflecting the reaction probability given the reaction conditions
k has units of reciprocal time (s-1)
-1 substrate

V = k[S1][S2] (second order reaction)
k has units of M-1 s-1
-2 substrates, more complicated

Question 9

Enzymatic Catalysis
-Enzymes do not affect
-Slow reactions face significant
-Enzymes increase
So a lower activation energy means a

Answer 9

-Enzymes do not affect equilibrium (ΔG)
-Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction
-Enzymes increase reaction rates (k) by decreasing ΔG‡

-So a lower activation energy means a faster rate

Question 10

Catalytic power: “Lock and Key” Model

Answer 10

Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP+, unbound and bound; another bound substrate, tetrahydrofolate, (PDB ID 1RA2). The NADP+binds to a pocket that is complementary to it in shape and ionic properties, an illustration of Emil Fischer’s “lock and key” hypothesis of enzyme action.

Question 11

ΔGB

Answer 11

ΔGB: Weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis

Question 12

binding energy

Answer 12

is the difference between the free energy that is catalyzed and not catalyzed-released by weak interactions
ex: hydrophobic

Question 13

Catalytic Mechanisms

Answer 13

-acid-base catalysis: give and take protons
-covalent catalysis: change reaction paths
-metal ion catalysis: use redox cofactors, pKa shifters
-electrostatic catalysis: preferential interactions with TS

Question 14

General acid-base catalysis
-Protons can be transferred to or from a
-Specific acid-base catalytic reactions use only the
-General acid-base catalysts use enzyme
Substrate and proton donors and proton acceptors are located

Answer 14

-Protons can be transferred to or from a charge species that favor breakdown to products instead of reactants

-Specific acid-base catalytic reactions use only the H3O+ or OH- ions present in water

-General acid-base catalysts use enzyme functional groups to assist in proton transfer reactions and facilitate bond cleavage

-Substrate and proton donors and proton acceptors are located side by side

Question 15

Covalent Catalysis

Answer 15

-A transient covalent bond between the enzyme and the substrate

-Changes the reaction Pathway

-Uncatalyzed: A—B —(H20)–> A+B

-Catalyzed: A—B+X:—>A—X+B—(H2O)-> A+X:+B

Requires a nucleophile on the enzyme
Can be a reactive serine, thiolate, amine, or carboxylate

Question 16

A transient covalent bond is formed between

Answer 16

A transient covalent bond is formed between enzyme and substrate

Question 17

Metal Ion Catalysis

Answer 17

-Involves a metal ion bound to the enzyme
-Interacts with substrate to facilitate binding
—-Stabilizes negative charges
-Participates in oxidation reactions
-Nearly a third of all enzymes require a metal ion for activity

Question 18

large hydrophobic groups bind easily to chymotrypsin. These are the only amino acids that can bind to it. Which are they?

Answer 18

(Phe, and Trp)
Phenylalanine
Tryptophan

Question 19

Zymogine

Answer 19

inactive form (original form) 1 single chain cut into 3 parts (small pieces) in order to be active and bind to substrate

Question 20

Role of Serine

Answer 20

functions as a nucleophile because of its hydroxyl group, it donates hydroxyl group to histidine–> (+)

Question 21

Role of histidine

Answer 21

functions as a general base, accepting a proton from other amino acid, thereby increasing its reactivity,

becomes stabilized by aspartic acid (-)

Question 22

Role of Asp

Answer 22

its negative charge stabilized the positive charge that develops on Histidine

Question 23

Steps in chymotrypsin

Answer 23

step 1: substrate binding
step 2:nucleophilic attack
step 3: substrate cleavage
Step 4: water comes in
step 5: water attacks
step 6: break-off from the enzyme
step 7:product dissociates

Question 24

What is enzyme kinetics?

Answer 24

-Kinetics is the study of the rate at which compounds react
-Rate of enzymatic reaction is affected by:
–enzyme
–substrate
–effectors
–temperature

Question 25

Why study enzyme kinetics?
-Quantitative description of
-Determine the order of
-Elucidate
-Understand
-Find effective
-Understand regulation of

Answer 25

-Quantitative description of biocatalysis
-Determine the order of binding of substrates
-Elucidate acid-base catalysis
-Understand catalytic mechanism
-Find effective inhibitors
-Understand regulation of activity

Question 26

How to Do Kinetic Measurements

Answer 26

Experiment:
1.Mix enzyme + substrate
2.Record rate of substrate disappearance/product formation as a function of time (the velocity (V) of reaction)
3.Plot initial velocity (V0) versus substrate concentration ([S]).
4.Change substrate concentration and repeat

-study relationship between reaction rate and the substrate that is being catalyzed

Question 27

Km

Answer 27

Km=Michaelis Constant-concentration when initial velocity is half of vmax

Question 28

high concentration substrate Increases

Answer 28

enzyme concentration becomes fixed

Question 29

input in research in relationship between concentration of substrate vs. velocity

Answer 29

Leonor Michaelis
Maud Menton

Question 30

Michaelis Menten Equation

Answer 30

Invertase Reaction:
sucrose + H2O glucose + fructose
V0= Vmax[S]/Km+[S]

Question 31

Km»[S],

Answer 31

[S] at denominator becomes insignificant numerator is significant. it will be proportional vmax (s)/ km

Question 32

[S]»Km

Answer 32

it becomes fully saturated so velocity doesn’t depend on substrate concentration. The (s) will cancel out and will V0=Vmax, you will be close or reach vmax.

Question 33

Burk-plot equation

Answer 33

1/V0= Km/Vmax[S] + 1/vmax

x-intertecpt is negative -1/km
y intercept 1/vmax

slope= km/vmax

Question 34

Intrinsic
extrinsic

Answer 34

intrinsic: does not depend on material (km)
Extrinsic: depends on material (vmax)

Question 35

Km is an ____ property

Answer 35

Intrinsic property of an enzyme- the bonding affinity between the substrate and enzyme
weak bond= large km
strong bond=small km

Question 36

Vmax is ___ property

Answer 36

extrinsic

Question 37

kcat =

Answer 37

kcat = Vmax/[Enz] (enzyme Conc.) What does kcat mean?

kcat comes from Vmax and [Enz]
Vmax is [molar]/sec [Enz] is in molar

Question 38

M-M Equation with kcat

Answer 38

The form of M-M equation for single substrate is

Vmax= Kcat [Etot]

kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second
Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme
Microscopic meaning of Km and kcat depends on the details of the mechanism

Question 39

Enzyme efficiency is limited by

Answer 39

diffusion:
kcat/KM
Enzyme reactions are diffusion-limited, can only work as fast as substrates gets to the active sites by molecular diffusion.

Question 40

Two-Substrate Reactions

Answer 40

Kinetic mechanism: the order of binding of substrates and release of products

When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms
Sequential mechanism
Ping-Pong mechanism

Question 41

Common mechanisms for enzyme-catalyzed bisubstrate reactions.

Answer 41

FIGURE 6–13 Common mechanisms for enzyme-catalyzed bisubstrate reactions. (a) The enzyme and both substrates come together to form a ternary complex. In ordered binding, substrate 1 must bind before substrate 2 can bind productively. In random binding, the substrates can
bind in either order. (b) An enzyme-substrate complex forms, a product leaves the complex, the altered enzyme forms a second complex with another substrate molecule, and the second product leaves, regenerating the enzyme. Substrate 1 may transfer a functional group to the enzyme (to form the covalently modified E’), which is subsequently transferred to substrate 2. This is called a Ping-Pong or double-displacement mechanism.

enzyme reaction involving a ternary complex:
-random order
-ordered

enzyme reaction in which no ternary complex is formed

Question 42

Sequential Kinetic Mechanism
We cannot easily
-Random mechanisms in equilibrium will give
-Lineweaver-Burk:

Answer 42

-We cannot easily distinguish random from ordered
-Random mechanisms in equilibrium will give intersection point at y-axis
-Lineweaver-Burk: lines intersect
Enzyme forming ternary complexes

Question 43

Ping-pong mechanism

Answer 43

Lineweaver-Burk: lines are parallel
Enzyme without ternary complexes

Question 44

Other approaches to understanding enzyme mechanism

Answer 44

-Structural information such as the 3-D structure- provided by crystallography
-Genetic engineering- look at how changes in specific amino acids affect enzyme structure and activity

Question 45

Enzyme Activity Inhibition: Inhibitors

Answer 45

Inhibitors are compounds that decrease enzyme’s activity

Irreversible inhibitors (inactivators) react with the enzyme
-One inhibitor molecule can permanently shut off one enzyme molecule
-They are often powerful toxins but also may be used as drugs

Reversible inhibitors bind to and can dissociate from the enzyme
-They are often structural analogs of substrates or products
-They are often used as drugs to slow down a specific enzyme

Reversible inhibitor can bind:
-to the free enzyme and prevent the binding of the substrate
-to the enzyme-substrate complex and prevent the reaction

Question 46

Competitive Inhibition

Answer 46

Competes with substrate for binding
-Binds active site
-Does not affect catalysis

-No change in Vmax; apparent increase in KM
-Lineweaver-Burk: lines intersect at the y-axis
-reverssible

Question 47

Uncompetitive Inhibition

Answer 47

-Only binds to ES complex
–Does not affect substrate binding
–Inhibits catalytic function

-Decrease in Vmax; apparent decrease in KM
-No change in KM/Vmax
-Lineweaver-Burk: lines are parallel
-reverssible

Question 48

Inhibitors - Mixed Inhibition

Answer 48

-Binds enzyme with or without substrate
–Binds to regulatory site
–Inhibits both substrate binding and catalysis
-Decrease in Vmax; apparent change in KM
-Lineweaver-Burk: lines intersect left from the y-axis
–Noncompetitive inhibitors are mixed inhibitors such that there is no change in KM (https://quizlet.com/68124133/uncompetitive-vs-competitive-vs-non-competitive-inhibition-flash-cards/)
-reversible

Question 49

Other Factors affecting enzyme activity

Answer 49

pH changes-
-amino acid side chains at the active site may be involved in reactions in which their ionization state is important
-pH optima varies from enzyme to enzyme but usually in range from 6-8
Temperature-
-At low temps, rate of reaction increases proportionally with increasing temp
-For many enzymes, the maximum rate is at 37ºC
-Above 50-60ºC rate falls off

Question 50

Enzyme activity can be regulated

Answer 50

Regulation (increased/decreased) can be:
-noncovalent modification
-covalent modification (e.g. phosphorylation)
-reversible (allosteric enzymes binding with regulatory compounds (modulator or effectors); enzymes bound by regulatory proteins)
-irreversible (peptide segments of enzymes removed by proteolytic cleavage)

Question 51

Regulatory Enzymes

Answer 51

-For a given metabolic pathway with multienzymes, the first enzyme in the sequence is regulatory – why?
-Un-necessary catalysis wastes energy and metabolites
-Generally the end product of a pathway will be inhibitory and this type of regulation is called feedback inhibition
-doesn’t have an inhibitor
-functions to regulate other enzyme’s activity
-have an active site and a regulatory site
-modulator binds to regulatory sites

Question 52

Feedback Inhibition is the Classic Form of Allosteric Inhibition

Answer 52

-Threonine dehydratase is inhibited by isoleucine- heterotrophic allosteric inhibition
-Isoleucine only binds to the regulatory site by noncovalent binding and is reversible. When the [isoleucine] decreases, the threonine dehydratase begins to work again

Question 53

Allosteric enzymes
-Regulation occurs as
-The regulatory site is
-If the modulator is the same as
-Heterotropic enzymes have
-Allosteric modulators are

Answer 53

-Regulation occurs as conformational changes of the modulator bind to convert different forms of the enzyme
-The regulatory site is specific for the modulator
-If the modulator is the same as the substrate then the enzyme is homotropic(substrate looks exactly like the modulator) and it is likely that the active site and regulatory site are the same
-Heterotropic enzymes have a modulator that is different than the substrate
-Allosteric modulators are not uncompetitive or mixed inhibitors

Question 54

Allosteric Effectors

Answer 54

– Bind to Allosteric Site
Allosteric enzymes are usually larger and more complex than nonallosteric enzymes

Question 55

Noncovalent Modification: Allosteric Regulators

Answer 55

Noncovalent Modification: Allosteric Regulators

The kinetics of allosteric regulators (Sigmoid kinetics) differ from Michaelis-Menten kinetics.

Question 56

Phosphorylation Affect Structure and Catalytic Activity of Enzymes

Answer 56

-Addition of phosphoryl group introduces a bulky charged group into a region
-The oxygen atoms of the phosphoryl group can be hydrogen bonded to several groups, commonly the amide groups of the peptide backbone.
-The two negative charges can repel other neighboring negatively charged groups

Question 57

Zymogens and activation
-they are activated by:

Answer 57

Zymogens (proenzymes) are inactive forms of enzymes
Zymogens are activated by removal of peptide sections, e.g. proinsulin is converted to insulin by removing a 33-amino acid peptide chain

-irreversible covalent modification
-activated when you cut peptide bonds