Transcript Section 5.3: Proteins - Clayton State University

Section 3
Proteins, Enzymes and
Central Metabolism
Chapter 5
Amino Acids, Peptides, &
Proteins
Section 5.1: Amino Acids
Figure 5.1 Protein
Diversity
Proteins are molecular tools
They are a diverse and complex group of
macromolecules
Section 5.1: Amino Acids
Proteins can be distinguished by the number,
composition, and sequence of amino acid residues
Amino acid polymers of 50 or less are peptides;
polymers greater than 50 are proteins or
polypeptides
There are 20 standard amino acids
Section 5.1: Amino Acids
Figure 5.3 General
Structure of the aAmino Acids
19 have the same general structure:
central (a) carbon, an amino group,
carboxylate group, hydrogen atom, and
an R group (proline is the exception)
At pH 7, the carboxyl group is in its
conjugate base form (-COO-) while the
amino group is its conjugate acid form (NH3+); therefore, it is amphoteric
Molecules that have both positive and
negative charges on different atoms are
zwitterions and have no net charge at
pH 7
The R group is what gives the amino
acid its unique properties
Section 5.1: Amino Acids
Figure 5.2 The
Standard Amino
Acids
Amino Acid Classes
Classified by their ability to interact with water
Nonpolar amino acids contain hydrocarbon groups
with no charge
Section 5.1: Amino Acids
Figure 5.2 The Standard Amino Acids
Amino Acid Classes Continued
Polar amino acids have functional groups that can
easily interact with water through hydrogen bonding
Contain a hydroxyl group (serine, threonine, and
tyrosine) or amide group (asparagine)
Section 5.1: Amino Acids
Figure 5.2 The
Standard Amino
Acids
Amino Acid Classes Continued
Acidic amino acids have side chains with a
carboxylate group that ionizes at physiological pH
Basic amino acids bear a positive charge at
physiological pH
At physiological pH, lysine is its conjugate acid
(-NH3+), arginine is permanently protonated, and
histidine is a weak base, because it is only partly
ionized
Section 5.1: Amino Acids
Section 5.1: Amino Acids
Biologically Active Amino Acids
Amino acids can have other biological
roles
1. Some amino acids or derivatives
can act as chemical messengers
Neurotransmitters (tryptophanderivative serotonin) and
hormones (tyrosine-derivative
thyroxine)
Figure 5.5 Some Derivatives of Amino Acids
Section 5.1: Amino Acids
2. Act as precursors for other
molecules (nucleotides and
heme)
3. Metabolic intermediates
(arginine, ornithine, and
citrulline in the urea cycle)
Figure 5.6 Citruline
and Ornithine
Section 5.1: Amino Acids
Figure 5.7 Modified
Amino Acid Residues
Found in Polypeptides
Modified Amino Acids in Proteins
Some proteins have amino acids that are modified
after synthesis
Serine, threonine, and tyrosine can be phosphorylated
g-Carboxyglutamate (prothtrombin), 4-hydroxyproline
(collagen), and 5-hydroxylysine (collagen)
Section 5.1: Amino Acids
Amino Acid Stereoisomers
Because the a-carbon (chiral carbon) is attached to
four different groups, they can exist as stereoisomers
Except glycine, which is the only nonchiral standard
amino acid
The molecules are mirror images
of one another, or enantiomers
They cannot be superimposed
over one another and rotate
plane, polarized light in opposite
directions (optical isomers)
Figure 5.8 Two Enantiomers
Section 5.1: Amino Acids
Figure 5.9 D- and L-Glyceraldehyde
Molecules are designated as D or L (glyceraldehyde is
the reference compound for optical isomers)
D or L is used to indicate the similarity of the
arrangement of atoms around the molecule’s
asymmetric carbon to the asymmetric carbon of the
glyceraldehyde isomers
Chirality has a profound effect on the structure and
function of proteins
Section 5.1: Amino Acids
Titration of Amino Acids
Free amino acids contain ionizable groups
The ionic form depends on the pH
When amino acids have no net charge due to
ionization of both groups, this is known as the
isoelectric point (pI) and can be calculated using:
pK1 + pK2
pI =
2
This formula only works if there is no pKR. If there is a pKR,
then you will need to determine which pK values are on either
side of zero net charge!
Section 5.1: Amino Acids
Section 5.1: Amino Acids
Alanine is a simple amino
acid with two ionizable
groups
Alanine loses two protons in
a stepwise fashion upon
titration with NaOH
Isoelectric point is reached
with deprotonation of the
carboxyl group
Figure 5.10 Titration of Two
Amino Acids: Alanine
Section 5.1: Amino Acids
pK1+pKR
2
Amino acids with ionizable side
chains have more complex titration
curves
= pKI
-2
-1
+1
0
Figure 5.10 Titration of Two
Amino Acids: Glutamic Acid
Glutamic acid is a good example,
because it has a carboxyl side chain
group
Glutamic acid has a +1 charge at
low pH
Glutamic acid’s isoelectric point
as base is added and the acarboxyl group loses a proton
As more base is added, it loses
protons to a final net charge of -2
Section 5.1: Amino Acids
Amino Acid Reactions
Amino acids, with their
carboxyl, amino, and various R
groups, can undergo many
chemical reactions
 Peptide bond and disulfide
bridge are of special interest
because of the effect they have
on structure
Figure 5.11 Formation of
a Dipeptide
Section 5.1: Amino Acids
Peptide Bond Formation:
polypeptides are linear
polymers of amino acids linked
by peptide bonds
Peptide bonds are amide
linkages formed by nucleophilic
acyl substitution
Dehydration reaction
Linkage of two amino acids is
a dipeptide
Figure 5.11 Formation of
a Dipeptide
Section 5.1: Amino Acids
Linus Pauling was the first to
characterize the peptide bond as
rigid and flat
Found that C-N bonds between
two amino acids are shorter than
other C-N bonds
Gives them partial doublebond characteristics (they are
resonance hybrids)
Because of the rigidity, one-third
of the bonds in a polypeptide
backbone cannot rotate freely
Limits the number of
conformational possibilities
Figure 5.12 The Peptide Bond
Section 5.1: Amino Acids
Cysteine oxidation leads to a
reversible disulfide bond
A disulfide bridge forms when
two cysteine residues form this
bond
Helps stabilize polypeptides
and proteins
Figure 5.13 Oxidation of
Two Cysteine Molecules
to Form Cystine
Section 5.2: Peptides
Less structurally complex than larger proteins,
peptides still have biologically important functions
Glutathione is a tripeptide found in most all
organisms and is involved in protein and DNA
synthesis, toxic substance metabolism, and amino
acid transport
Vasopressin is an antidiuretic hormone that
regulates water balance, appetite, and body
temperature
Oxytocin is a peptide that aids in uterine
contraction and lactation
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Of all the molecules in a living organism, proteins
have the most diverse set of functions:
Catalysis (enzymes)
Structure (cell and organismal)
Movement (amoeboid movement)
Defense (antibodies)
Regulation (insulin is a peptide hormone)
Transport (membrane transporters)
Storage (ovalbumin in bird eggs)
Stress Response (heat shock proteins)
Section 5.3: Proteins
Due to recent research, numerous multifunction
proteins have been identified
Proteins are categorized into families based on
sequence and three-dimensional shape
Superfamilies are more distantly related proteins
(e.g., hemoglobin and myoglobin to neuroglobin)
Proteins are also classified by shape: globular and
fibrous
Proteins can be classified by composition: simple
(contain only amino acids) or conjugated
Conjugated proteins have a protein and nonprotein
component (i.e., lipoprotein or glycoprotein)
Section 5.3: Proteins
Protein Structure
Proteins are extraordinarily
complex; therefore, simpler images
highlighting specific features are
useful
Space-filling and ribbon models
Levels of protein structure are
primary, secondary, tertiary, and
quaternary
Figure 5.15 The Enzyme
Adenylate Kinase
Section 5.3: Proteins
Figure 5.16 Segments of b-chain in HbA and HbS
Primary Structure is the specific amino acid
sequence of a protein
Homologous proteins share a similar sequence and
arose from the same ancestor gene
When comparing amino acid sequences of a protein
between species, those that are identical are invariant
and presumed to be essential for function
Section 5.3: Proteins
Secondary Structure: Polypeptide
secondary structure has a variety of
repeating structures
Figure 5.18 The a-Helix
Most common include the a-helix and bpleated sheet
Both structures are stabilized by hydrogen
bonding between the carbonyl and the N-H
groups of the polypeptide’s backbone
The a-helix is a rigid, rod-like structure
formed by a right-handed helical turn
a-Helix is stabilized by N-H hydrogen
bonding with a carbonyl four amino acids
away
Glycine and proline do not foster
a-helical formation
Section 5.3: Proteins
Figure 5.19 b-Pleated
Sheet
The b-pleated sheets form when two or more
polypeptide chain segments line up, side by side
Section 5.3: Proteins
Each b strand is fully
extended and stabilized by
hydrogen bonding between
N-H and carbonyl groups of
adjacent strands
Parallel sheets are much
less stable than
antiparallel sheets
Figure 5.19 b-Pleated Sheet
Section 5.3: Proteins
Figure 5.20 Selected Supersecondary Structures
Many proteins form supersecondary structures
(motifs) with patterns of a-helix and b-sheet structures
(a) bab unit
(b) b-meander
(c) aa unit
(d) b-barrel
(e) Greek key
Section 5.3: Proteins
Tertiary Structure refers to unique threedimensional structures formed by globular proteins
Also prosthetic groups
Protein folding is the process by which a nascent
molecule acquires a highly organized structure
Information for folding is contained within the
amino acid sequence
Interactions of the side chains are stabilized by
electrostatic forces
Tertiary structure has several important features
1. Many polypeptides fold in a way to bring distant amino
acids into close proximity
2. Globular proteins are compact because of efficient packing
Section 5.3: Proteins
Tertiary structure has several important features
1. Many polypeptides fold in a way to bring distant amino acids
into close proximity
2. Globular proteins are compact because of efficient packing
3. Large globular proteins (200+ amino acids) often contain
several domains
Domains are structurally independent segments that have
specific functions
Core structural element of a domain is called a fold
4. A number of proteins called mosaic or modular proteins consist
of repeated domains
Fibronectin has three repeated domains (F1, F2, and F3)
Domain modules are coded for by genetic sequences
created by gene duplications
Section 5.3: Proteins
Figure 5.21 Selected Domains Found in Large Numbers of Proteins
Section 5.3: Proteins
Figure 5.23 Interactions
That Maintain Tertiary
Structure
Interactions that stabilize tertiary structure are
hydrophobic interactions, electrostatic interactions
(salt bridges), hydrogen bonds, covalent bonds, and
hydration
Section 5.3: Proteins
Figure 5.25 Structure of
Immunoglobulin G
Quaternary structure: a protein that is composed of
several polypeptide chains (subunits)
Multisubunit proteins may be composed, at least in
part, of identical subunits and are referred to as
oligomers (composed of protomers)
Section 5.3: Proteins
Reasons for common occurrence of
multisubunit proteins:
1. Synthesis of subunits may be
more efficient
2. In supramolecular complexes
replacement of worn-out
components can be handled
more effectively
3. Biological function may be
regulated by complex
interactions of multiple
subunits
Figure 5.25 Structure of
Immunoglobulin G
Section 5.3: Proteins
Polypeptide subunits held together
with noncovalent interactions
Covalent interactions like
disulfide bridges
(immunoglobulins) are less
common
Other covalent interactions
include desmosine and
lysinonorleucine linkages
Figure 5.26 Desmosine
and Lysinonorleucine
linkages
Section 5.3: Proteins
Interactions between subunits are often affected by
ligand binding
An example of this is allostery, which controls protein
function by ligand binding
Can change protein conformation and function
(allosteric transition)
Ligands triggering these transitions are effectors
and modulators
Section 5.3: Proteins
Figure 5.27 Disordered
Protein Binding
Unstructured proteins: Some proteins are partially
or completely unstructured
Unstructured proteins referred to as intrinsically
unstructured proteins (IUPs) or natively unfolded
proteins
Often these proteins are involved in searching out
binding partners (i.e., KID domain of CREB)
Section 5.3: Proteins
Figure 5.28 The Anfinsen
Experiment
Loss of Protein Structure: Because of small differences
between the free energy of folded and unfolded proteins,
they are susceptible to changes in environmental factors
Disruption of protein structure is denaturation (reverse is
renaturation)
Denaturation does not disrupt primary protein structure
Section 5.3: Proteins
The Folding Problem
The direct relationship between a protein’s primary
sequence and its final three-dimensional conformation
is among the most important assumptions in
biochemistry
Painstaking work has been done to be able to predict
structure by understanding the physical and chemical
properties of amino acids
X-ray crystallography, NMR spectroscopy, and sitedirected mutagenesis
Section 5.3: Proteins
Important advances have
been made by biochemists in
protein-folding research
This research led to the
understanding that it is
not a single pathway
A funnel shape best
describes how an unfolded
protein negotiates its way to a
low-energy, folded state
Numerous routes and
intermediates
Figure 5.29 The Energy Landscape
for Protein Folding
Section 5.3: Proteins
Small polypeptides (<100
amino acids) often form
with no intermediates
Larger polypeptides often
require several
intermediates (molten
globules)
Many proteins use
molecular chaperones to
help with folding and
targeting
Figure 5.30 Protein Folding
Section 5.3: Proteins
Molecular chaperones assist
protein folding in two ways:
Preventing inappropriate
protein-protein interactions
Helping folding occur rapidly
and precisely
Two major classes: Hsp70s and
Hsp60s (chaperonins)
Figure 5.31 Space-Filling
Model of the E. Coli
Chaperonin, called the
GroES-GroEL Complex
Section 5.3: Proteins
Hsp70s are a family of
chaperones that bind and
stabilize proteins during the
early stages of folding
Hsp60s (chaperonins) mediate
protein folding after the protein
is released by Hsp70
Increases speed and
efficiency of the folding
process
Both use ATP hydrolysis
Both are also involved in
refolding proteins
If refolding is not possible,
molecular chaperones promote
protein degradation
Figure 5.32 The Molecular Chaperones
Section 5.3: Proteins
Fibrous Proteins
Typically contain high
proportions of a-helices
and b-pleated sheets
Often have structural
rather than dynamic
roles and are water
insoluble
Keratin (a-helices) and
silk fibroin (b-sheets)
Figure 5.33 a-Keratin
Section 5.3: Proteins
Globular Proteins
Biological functions often include
precise binding of ligands
Myoglobin and hemoglobin
Both have a specialized heme
prosthetic group used for
reversible oxygen binding
Figure 5.36 Heme
Section 5.3: Proteins
Myoglobin: found in high
concentrations in cardiac
and skeletal muscle
The protein component
of myoglobin, globin, is a
single protein with eight
a-helices
Encloses a heme [Fe2+]
that has a high affinity
for O2
Figure 5.37 Myoglobin
Section 5.3: Proteins
Hemoglobin is a roughly spherical
protein found in red blood cells
Figure 5.38 The OxygenBinding Site of Heme
Created by a Folded
Globin Chain
Primary function is to transport
oxygen from the lungs to tissues
HbA molecule is composed of 2
a-chains and 2 b-chains (a2b2)
2% of hemoglobin contains dchains instead of b-chains (HbA2)
Embryonic and fetal hemoglobin
have e- and g-chains that have a
higher affinity for O2
Section 5.3: Proteins
Figure 5.39 Hemoglobin
Comparison of myoglobin and hemoglobin identified
several invariant residues, most having to do with
oxygen binding
Four chains of hemoglobin arranged as two identical
ab dimers
Section 5.3: Proteins
Figure 5.41 Equilibrium
Curves Measure the
Affinity of Hemoglobin and
Myoglobin for Oxygen
Hemoglobin shows a sigmoidal oxygen dissociation
curve due to cooperative binding
Binding of first O2 changes hemoglobin’s
conformation making binding of additional O2 easier
Myoglobin dissociation curve is a hyperbolic simpler
binding pattern
Section 5.3: Proteins
Binding of ligands other than oxygen affects
hemoglobin’s oxygen-binding properties
pH decrease enhances oxygen release from
hemoglobin (Bohr effect)
The waste product CO2 also enhances oxygen
release by increasing H+ concentration
Binding of H+ to several ionizable groups on
hemoglobin shifts it to its T state
Section 5.3: Proteins
Figure 5.42 The Effect of 2,3Bisphosphoglycerate (BPG) on
the Affinity Between Oxygen
and Hemoglobin
2,3-Bisphosphoglycerate (BPG) is also an important
regulator of hemoglobin function
Red blood cells have a high concentration of BPG,
which lowers hemoglobin’s affinity for O2
In the lungs, these processes reverse
Section 5.4: Molecular Machines
Molecular Machines
Purposeful movement is a hallmark of living things
This behavior takes a myriad of forms
Biological machines are responsible for these
behaviors
Usually ATP or GTP driven
Motor proteins fall into the following categories:
1. Classical motors (myosins, dyneins, and
kinesin)
2. Timing devices (EF-Tu in translation)
3. Microprocessing switching devices (G proteins)
4. Assembly and disassembly factors (cytoskeleton
assembly and disassembly)
Chapter 6
Enzymes
Section 6.1: Properties of Enzymes
A chemical reaction occurs when colliding molecules
possess a minimum amount of energy called the
activation energy (Ea)
More commonly called free energy of activation (DG‡)
in biochemistry
Many reactions that are spontaneous (-DG) will
proceed at imperceptibly slow rates, because they do
not have the energy or correct orientation
The likelihood of a reaction improves with increasing
the temperature or using a metal catalyst
Section 6.1: Properties of Enzymes
Living systems cannot increase temperature without
the risk of damaging structures, so they use catalysts
(enzymes)
Enzymes can increase reaction rate up to 107 to 1019
Enzymes are also very specific for substrates
Section 6.1: Properties of Enzymes
Figure 6.1 A Catalyst Reduces the
Activation Energy of a Reaction
Catalysts increase reaction rate by lowering
activation energy
The free energy of activation (DG‡) is the amount of
energy to convert 1 mol of substrate (reactant) from
the ground state to the transition state
Section 6.1: Properties of Enzymes
Each enzyme has a specific active site to bind the
substrate
The active site also has amino acid side chains that
take an active role in the catalytic process
The active site is used to optimally orient the
substrate to achieve the transition state at a lower
energy
Section 6.1: Properties of Enzymes
Figure 6.2 The Induced
Fit Model
Two models that describe enzyme binding of
substrate:
Lock and key and induced fit
Some enzymes require certain non-protein
components to function: cofactors and coenzymes
Intact functional enzymes with cofactors are
holoenzymes
The protein component is the apoenzyme
Section 6.2: Classification of Enzymes
International Union of Biochemistry (IUB)
instituted a naming convention for enzymes, based
upon the type of chemical reaction catalyzed
Six major enzyme categories:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
Section 6.3: Enzyme Kinetics
Thermodynamics can predict whether a reaction is
spontaneous, but cannot predict rate
The rate or velocity of a reaction is the change of a
concentration of reactant or product per unit of time
Section 6.3: Enzyme Kinetics
Initial velocity (v0) is a velocity at
the beginning of a reaction when
the concentration of substrate
greatly exceeds enzyme
concentration
Information about reaction rates
is the quantitative study of enzyme
catalysis, or enzyme kinetics
Figure 6.3a Enzyme
Kinetic Studies
Section 6.3: Enzyme Kinetics
Kinetics also measures enzyme
affinity for substrates and inhibitors
Order is useful in describing
reactions; it is determined
experimentally
First order is unimolecular (no
collisions required)
Rate = k[A]1
Figure 6.3b Enzyme
Kinetic Studies
Half-life is the time for one-half of
the reactant molecules to be
consumed
Section 6.3: Enzyme Kinetics
Second order is bimolecular (A + B
 P)
Rate = k[A]1[B]1
When a reaction is zero order, the
rate is not affected by adding more
substrate
Enzyme substrate sites saturated
Figure 6.3 Enzyme Kinetic Studies
Section 6.3: Enzyme Kinetics
Michaelis-Menten Kinetics
The concept of enzyme substrate complexes:
E+S
k1
k-1
ES
k2
E+P
Introduce the Michaelis constant Km
k-1 + k2
Km =
k1
When Km is experimentally determined, it is a
constant that is characteristic of the enzyme and the
substrate under specific conditions
The lower the value of Km, the greater the affinity
of the enzyme for ES complex formation
Section 6.3: Enzyme Kinetics
Vmax is the maximum velocity a
reaction can attain
The number of substrate
molecules converted to product
per unit time is kcat
kcat is Vmax over total enzyme
concentration (Et)
Figure 6.4 Initial Reaction
Velocity v0 and Substrate
Concentration [S] for a
Typical Enzyme-Catalyzed
Reaction
ν=
Vmax[S]
[S] + Km
Michaelis-Menten Equation
Section 6.3: Enzyme Kinetics
Figure 6.5 A Michaelis-Menten Plot
The specificity constant
reflects the relationship
between catalytic rate and
substrate binding affinity
(kcat/Km)
Specific activity is a measure
used to identify enzyme
purification
Section 6.3: Enzyme Kinetics
Section 6.3: Enzyme Kinetics
Lineweaver-Burk Plots
Using the reciprocal of the
Michaelis-Menten equation
obtains a more accurate
determination of the values
Figure 6.6 Lineweaver-Burk or
Double-Reciprocal Plot
Slope of the line Km/Vmax
1/Vmax is the Y intercept
-1/Km is the X intercept
Section 6.3: Enzyme Kinetics
Multisubstrate Reactions
Most reactions involve two or more substrates in
two classes:
Sequential—reaction cannot proceed until all
substrates are bound to the enzyme active site
Ordered and random
Double-Displacement Reactions—first product is
released before second substrate binds
Enzyme is altered by first phase of the reaction
Section 6.3: Enzyme Kinetics
Enzyme Inhibition
Inhibitors reduce enzyme activity
In living systems inhibitors are important, because
they regulate metabolic pathways
Enzyme inhibition can be reversible or irreversible:
Reversible inhibition can be counteracted by
increasing substrate levels or removing the inhibitor
Competitive, noncompetitive, and uncompetitive
Irreversible inhibition occurs when the inhibitor
permanently impairs the enzyme (covalent
interaction)
Section 6.3: Enzyme Kinetics
Competitive Inhibitors bind
reversibly to the enzyme at the
active site, thus competing
with substrate binding
Figure 6.8 Michaelis-Menten
Plot of Uninhibited Enzyme
Activity Versus Competitive
Inhibition
Forms enzyme-inhibitor (EI)
complex
Increasing substrate
concentration overcomes
competitive inhibition
Section 6.3: Enzyme Kinetics
Noncompetitive Inhibitors can
bind reversibly to the ES
complex at a site other than
the active site
Figure 6.10 MichaelisMenten Plot of Uninhibited
Enzyme Activity Versus
Noncompetitive Inhibition
Forms EI + S and EIS
complex
Changes enzyme
conformation
Increased substrate
concentration partially
reverses inhibition
This is the case for pure
noncompetitive inhibition only
Section 6.3: Enzyme Kinetics
Uncompetitive Inhibitors: a type of uncompetitive
inhibition that involves binding only after substrate
is bound
Ineffective at low substrate concentrations
Kinetic Analysis of Enzyme Inhibition: doublereciprocal plots may be used to distinguish
competitive, noncompetitive, and uncompetitive
inhibition
Section 6.3: Enzyme Kinetics
Figure 6.11 Kinetic
Analysis of Enzyme
Inhibition
Competitive inhibition increases Km, not Vmax (6.10a)
Pure noncompetitive Vmax lowered Km unchanged
(6.10b)
Section 6.3: Enzyme Kinetics
Figure 6.11
Kinetic Analysis
of Enzyme
Inhibition
Mixed noncompetitive inhibition—both Vmax and Km
change and intersection occur above or below the
horizontal axis due to differences in k values (6.10c & d)
Section 6.3: Enzyme Kinetics
Figure 6.11
Kinetic Analysis
of Enzyme
Inhibition
Uncompetitive—Km and Vmax are changed although
ratio is the same (6.10e)
Section 6.3: Enzyme Kinetics
Allosteric Enzymes have a
sigmoidal curve rather than a
hyperbolic one
Figure 6.13 The Kinetic Profile
of an Allosteric Enzyme
Resembles the oxygen-binding
curve of hemoglobin
Michaelis-Menten kinetics do
not apply to allosteric enzymes
Section 6.3: Enzyme Kinetics
Enzyme Kinetics, Metabolism, and Macromolecular
Crowding
Ultimate goal is understanding enzyme kinetics in
living organisms
In vitro work does not always reflect in vivo reality
Cell shows macromolecular crowding, which
influences reaction rates and equilibrium constants
Systems biologists are using computer modeling, in
vitro, and in vivo data to overcome issues
Section 6.4: Catalysis
Scientists use X-ray crystallography, chemical
inactivation, and modeling to understand the
catalytic mechanism of enzymes
Organic Reactions and the Transition State
Essential features are the reaction between
electron-deficient atoms (electrophiles) and electronrich atoms (nucleophiles)
A reaction mechanism is a step-by-step description
of a reaction
Electrons flow from a nucleophile to an electrophile
Section 6.4: Catalysis
Figure 6.14 Energy Profile
for a Two-Step Reaction
One or more intermediates may form during the
course of a reaction
Examples of reactive intermediates include free
radicals, carbocations, and carbanions
Section 6.4: Catalysis
Figure 6.14 Energy Profile
for a Two-Step Reaction
In any reaction, only molecules that reach the
transition state can convert into product molecules
Stabilizing the transition state lowers energy of
activation (Ea) and increases reaction rate
Section 6.4: Catalysis
Catalytic Mechanisms
Mechanisms of only a few enzymes are known in
significant detail
Several factors contribute to enzyme catalysis. The
most important are:
Proximity and Strain Effects—the substrate must
come in close proximity to the active site
Electrostatic Effects—charge distribution in the
largely anhydrous active site may help position the
substrate
Section 6.4: Catalysis
Figure 6.15 Ester Hydrolysis:
Hydroxide Ion Catalysis
Acid-Base Catalysis—proton transfer is an important
factor in chemical reactions
Hydrolysis of an ester, for example, takes place better
if the pH is raised
Hydroxide ion catalysis
Section 6.4: Catalysis
Figure 6.15 Ester Hydrolysis:
General Base Catalysis
More physiological is the use of general bases and
acids
Side chains of many amino acids (e.g., histidine, lysine,
and aspartate) can be used as general acids or bases
Depends on state of protonation, based on pKa of
functional groups
Section 6.4: Catalysis
Figure 6.15 Ester Hydrolysis:
General Acid Catalysis
Covalent Catalysis—the formation of an unstable
covalent bond with a nucleophilic group on the
enzyme and an electrophilic group on the substrate
Section 6.4: Catalysis
The Roles of Amino Acids in Enzyme Catalysis
The active sites of enzymes are lined with amino
acids that create a microenvironment conducive to
catalysis
Residues can be catalytic or noncatalytic
In order to participate in catalysis, the amino acid
has to be charged or polar
For example, chymotrypsin action in Figure 6.16
Noncatalytic side groups function to orient substrate
or stabilize transition state
Section 6.4: Catalysis
The Role of Cofactors in Enzyme Catalysis
Many proteins require nonprotein cofactors
Metals—important metals in living organisms are
alkali metals (Na+, K+, Mg2+, and Ca2+) and transition
metals (Zn2+, Fe2+, and Cu2+)
Alkali metals are usually loosely bound and play
structural roles
Transition metals usually play a functional role in
catalysis as part of a functional group
Metals are good Lewis acids and effective electrophiles
Section 6.4: Catalysis
Coenzymes—a group of organic molecules that
provide enzymes’ chemical versatility
Contain functional groups that amino acid side
chains do not
Can be tightly or loosely bound and their structures
are often changed by the catalytic process
Most are derived from vitamins
Three groups: electron transfer (NAD+), group
transfer (coenzyme A), and high-energy transfer
potential (nucleotides)
Section 6.4: Catalysis
Effects of Temperature and pH
on Enzyme-Catalyzed Reactions
Change in an environmental
factor could change enzyme
structure and therefore function
Temperature—the higher the
temperature, the faster the
reaction rate; increased number
of collisions
Figure 6.16 The Effect of
Temperature on Enzyme
Activity
Enzymes are proteins and
become denatured at high
temperatures
Section 6.4: Catalysis
Figure 6.17 The Effect of pH
on Two Enzymes
pH—hydrogen ion concentration affects enzyme
function; therefore, there is a pH optimum
Catalytic activity is related to ionic state of the active
site
Changes in ionizable groups could change structure of
the enzyme
Section 6.4: Catalysis
Detailed Mechanisms of Enzyme Catalysis
Mechanisms of two well-characterized enzymes:
Chymotrypsin—serine protease of 27,000 D
Serine proteases have a triad of amino acids in their
active site (e.g., Asp 102, His 57, and Ser 195)
Hydrolyzes peptide bonds adjacent to aromatic amino
acids
Section 6.4: Catalysis
Figure 6.18 The Probable
Mechanism of Action of
Chymotrypsin
Section 6.4: Catalysis
Figure 6.18 The Probable
Mechanism of Action of
Chymotrypsin
Section 6.5: Enzyme Regulation
Figure 6.19 Alcohol
Dehydrogenase
Alcohol Dehydrogenase—catalyzes the reversible
oxidation of alcohols to aldehydes or ketones
Uses NAD+ as a hydride (H:-) ion acceptor
Section 6.5: Enzyme Regulation
Enzyme regulation is necessary for:
Maintenance of ordered state
Conservation of energy
Responsiveness to environmental changes
Control is accomplished by genetic control, covalent
modification (e.g. phosphorylation) , allosteric
regulation, and compartmentation
Section 6.5: Enzyme Regulation
Genetic Control
Genetic control plays an important role in
controlling the synthesis of enzymes
Happens at the DNA level and can lead to repression
or induction of enzyme synthesis
Section 6.5: Enzyme Regulation
Covalent Modification
Figure 6.20 The Activation
of Chymotrypsinogen
Several covalent modifications
in enzyme structure cause
changes in function
Types of covalent modification
include phosphorylation,
methylation, acetylation, and
nucleotidylation
Some enzymes produced and
stored as proenzymes or
zymogens
Section 6.5: Enzyme Regulation
Allosteric Regulation
Figure 6.21 The Rate of an
Enzyme-Catalyzed Reaction
as a Function of Substrate
Concentration
Enzymes that are regulated
by the binding of effectors at
allosteric sites
Sigmoidal curve, unlike
Michaelis-Menten kinetics
If the effectors are substrates,
then it is homotropic; if the
ligand is different, then it is
heterotropic
Section 6.5: Enzyme Regulation
Figure 6.22a Allosteric
Interaction Models
Most allosteric enzymes are multisubunit enzymes
Two theoretical models: concerted and sequential
In the concerted model, all subunits are changed at
once from taut (T) to relaxed (R) or vice versa
An activator shifts the equilibrium in favor of the R
form; an inhibitor shifts in favor of the T form
Section 6.5: Enzyme Regulation
Figure 6.22b Allosteric Interaction Models
Concerted model is supported by positive
cooperativity where binding of one ligand increases
subsequent binding
It is not supported by negative cooperativity
Section 6.5: Enzyme Regulation
Figure 6.22 Allosteric
Interaction Models
In the sequential model binding of the ligand to one
subunit, it triggers a conformational change that is
passed to subsequent subunits
A more complex model that allows for intermediate
formations
Accounts for both positive and negative cooperativity
Neither model perfectly accounts for all enzyme
behavior
Section 6.5: Enzyme Regulation
Compartmentation
Compartments created by cellular infrastructure
regulate biochemical reactions
Physical separation makes separate control possible
Solves several problems:
Divide and control
Diffusion barriers
Specialized reaction conditions
Damage control
Chapter 8
Carbohydrate Metabolism
Metabolism and Jet Engines
Catabolic pathways
with a turbo step are
optimized and
efficient
Energy is fed back
into the system to
accelerate the fuel
input step
Figure 8.1 Glycolysis and the Turbo Jet Engine
Chapter 8: Overview
Figure 8.2 Major Pathways in
Carbohydrate Metabolism
Energy transforming pathways of carbohydrate
metabolism include glycolysis, glycogenesis,
glycogenolysis, gluconeogenesis, and pentose phosphate
pathway
Section 8.1: Glycolysis
Figure 8.2 Major Pathways in
Carbohydrate Metabolism
Glycolysis (anaerobic process) occurs in almost
every living cell
Ancient process central to all life
Splits glucose into two three-carbon pyruvate units
Catabolic process that captures some energy as
2 ATP and 2 NADH
Section 8.1: Glycolysis
Glycolysis is an anaerobic process
Two stages (stage 1 and 2): energy investment and
energy producing
Glycolytic Pathway: D-Glucose + 2 ADP + 2 Pi + 2
NAD+  2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2
H 2O
Section 8.1: Glycolysis
Figure 8.3 Glycolytic Pathway
Section 8.1: Glycolysis
Figure 8.3 Glycolytic
Pathway
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway
1. Synthesis of glucose-6phosphate
Phosphorylation of
glucose (kinase) prevents
transport out of the cell
and increases reactivity
2. Conversion of glucose-6phosphate to fructose-6phosphate
Figure 8.3a Glycolytic Pathway
Conversion of aldose to
ketose
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
3. Phosphorylation of
fructose-6-phosphate
This step is irreversible
due to a large decrease in
free energy and commits
the molecule to glycolysis
4. Cleavage of fructose-1,6bisphosphate
Figure 8.3a Glycolytic Pathway
Aldol cleavage giving an
aldose and ketose product
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
5. Interconversion of
glyceraldehyde-3phosphate and
dihydroxyacetone
phosphate
Conversion of aldose to
ketose enables all carbons
to continue through
glycolysis
Figure 8.3a Glycolytic Pathway
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
In Step 2 (reactions 6-10), each
reaction occurs in duplicate
6. Oxidation of glyceraldehyde3-phosphate
Creates high-energy
phosphoanhydride bond for
ATP formation and NADH
7. Phosphoryl group transfer
Production of ATP via
substrate-level
phosphorylation
Figure 8.3b Glycolytic Pathway (Stage 2)
Section 8.1: Glycolysis
Figure 8.4Glyceraldehyde-3-Phosphate
Dehydrogenase Reaction
Oxidation of glyceraldehyde-3-phosphate (G-3-P) is a
2-step process (reaction 6)
G-3-P undergoes oxidation and phosphorylation
G-3-P interacts with the sulfhydryl group in the
enzyme’s active site
Section 8.1: Glycolysis
Figure 8.4 Glyceraldehyde-3-Phosphate
Dehydrogenase Reaction
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
7. Phosphoryl group transfer
Production of ATP via
substrate-level
phosphorylation
Figure 8.3b Glycolytic Pathway (Stage 2)
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
8. Interconversion of
3-phosphoglycerate and
2-phosphoglycerate
First step in formation of
phosphoenolpyruvate (PEP)
9. Dehydration of
2-phosphoglycerate
Production of PEP, which has
a high phosphoryl group
transfer potential
(tautomerization), locks it into
the highest energy form
Figure 8.4b Glycolytic Pathway (Stage 2)
Section 8.1: Glycolysis
Reactions of the Glycolytic
Pathway Continued
10. Synthesis of pyruvate
Formation of pyruvate and ATP
Produces a net of 2 ATP, 2
NADH, and 2 pyruvate
Figure 8.3b Glycolytic Pathway (Stage 2)
Section 8.1: Glycolysis
Oxidation of glyceraldehyde-3-phosphate (G-3-P) is
a complex process (reaction 6)
Substrate oxidized after interaction with sulfhydryl
Bound NADH exchanged for NAD+
Enzyme displaced by addition of inorganic
phosphate
Section 8.1: Glycolysis
Figure 8.6 The Fates of Pyruvate
The Fates of Pyruvate
Pyruvate is an energy-rich molecule
Under aerobic conditions, pyruvate is converted to
acetyl-CoA for use in the citric acid cycle and electron
transport chain
Section 8.1: Glycolysis
The Fates of Pyruvate
Continued
Under anaerobic conditions
pyruvate can undergo
fermentation: alcoholic or
homolactic
Regenerates NAD+ so
glycolysis can continue
Figure 8.7 Recycling NADH
during Anaerobic Glycolysis
Section 8.1: Glycolysis
Figure 8.8 Free Energy Changes during Glycolysis in Red Blood Cells
Energetics of Glycolysis
In red blood cells, only three reactions have
significantly negative DG values
Section 8.1: Glycolysis
Regulation of Glycolysis
The rate of the glycolytic pathway in a cell is
controlled by the allosteric enzymes:
Hexokinases I, II, and III
PFK-1
Pyruvate kinase
Allosteric enzymes are sensitive indicators of a cell’s
metabolic state regulated locally by effector molecules
The peptide hormones glucagon and insulin also regulate
glycolysis
Section 8.1: Glycolysis
Regulation of Glycolysis Continued
High AMP concentrations activate pyruvate kinase
Fructose-2,6-bisphosphate, produced via hormoneinduced covalent modification of PFK-2, activates
PFK-1
Accumulation of fructose-1,6-bisphosphate activates
PFK-1 providing a feed-forward mechanism
Section 8.1: Glycolysis
Figure 8.9 Fructose-2,6-Bisphosphate Level Regulation
Section 8.2: Gluconeogenesis
Gluconeogenesis is the formation of new glucose
molecules from precursors in the liver
Precursor molecules include lactate, pyruvate, and
a-keto acids
Gluconeogenesis Reactions
Reverse of glycolysis except the three irreversible
reactions
Section 8.2: Gluconeogenesis
Figure 8.10 Carbohydrate Metabolism: Gluconeogenesis and Glycolysis
Section 8.2: Gluconeogenesis
Figure 8.10 Carbohydrate Metabolism: Gluconeogenesis and Glycolysis
Section 8.2: Gluconeogenesis
Gluconeogenesis Reactions Continued
Three bypass reactions:
1. Synthesis of phosphoenolpyruvate (PEP) via the
enzymes pyruvate carboxylase and pyruvate
carboxykinase
2. Conversion of fructose-1,6-bisphosphate to fructose6-phosphate via the enzyme fructose-1,6bisphosphatase
3. Formation of glucose from glucose-6-phosphate via
the liver and kidney-specific enzyme glucose-6phosphatase
Section 8.2: Gluconeogenesis
Gluconeogenesis Substrates
Three of the most important
substrates for gluconeogenesis
are:
1. Lactate—released by
skeletal muscle from the Cori
cycle
After transfer to the liver
lactate is converted to
pyruvate, then to glucose
2. Glycerol—a product of fat
metabolism
Figure 8.11 Cori Cycle
Section 8.2: Gluconeogenesis
Figure 8.12 The Glucose Alanine Cycle
Gluconeogenesis Substrates Continued
3. Alanine—generated from pyruvate in exercising
muscle
Alanine is converted to pyruvate and then glucose
in the liver
Section 8.2: Gluconeogenesis
Gluconeogenesis
Regulation
Substrate availability
Hormones (e.g., cortisol
and insulin)
Figure 8.13 Allosteric Regulation of
Glycolysis and Gluconeogenesis
Section 8.2: Gluconeogenesis
Gluconeogenesis
Regulation Continued
Allosteric enzymes
(pyruvate carboxylase,
pyruvate
carboxykinase,
fructose-1,6bisphosphatase, and
glucose-6-phosphatase)
+
Figure 8.13 Allosteric Regulation of
Glycolysis and Gluconeogenesis
Section 8.3: Pentose Phosphate Pathway
Glucose-6-phosphate
dehydrogenase
Gluconolactonase
Pentose Phosphate
Pathway
Alternate glucose
metabolic pathway
Products are NADPH
and ribose-5phosphate
Two phases:
oxidative and
nonoxidative
Figure 8.14a The Pentose Phosphate Pathway (oxidative)
Section 8.3: Pentose Phosphate Pathway
6-phosphogluconate
dehydrogenase
Pentose Phosphate
Pathway: Oxidative
Three reactions
Results in ribulose5-phosphate and two
NADPH
NADPH is a
reducing agent used
in anabolic processes
Figure 8.14a The Pentose Phosphate Pathway (oxidative)
Section 8.3: Pentose Phosphate Pathway
Pentose Phosphate
Pathway: Nonoxidative
Produces important
intermediates for nucleotide
biosynthesis and glycolysis
Ribose-5-phosphate
Glyceraldehyde-3phosphate
Fructose-6-phosphate
Figure 8.14b The Pentose Phosphate
Pathway (nonoxidative)
Section 8.3: Pentose Phosphate Pathway
Pentose Phosphate
Pathway
If the cell requires
more NADPH than
ribose molecules,
products of the
nonoxidative phase
can be shuttled into
glycolysis
Figure 8.15 Carbohydrate
Metabolism: Glycolysis
and the Phosphate
Pathway
Section 8.4: Metabolism of Other Important Sugars
Figure 8.16 Carbohydrate Metabolism:
Galactose Metabolism
Fructose, mannose, and galactose are also important
sugars for vertebrates
Most common sugars found in oligosaccharides
besides glucose
Section 8.4: Metabolism of Other Important Sugars
Fructose Metabolism
Second to glucose in the human diet
Can enter the glycolytic pathway in two ways:
Through the liver (multi-enzymatic process)
Muscle and adipose tissue (hexokinase)
Section 8.4: Metabolism of Other Important Sugars
Figure 8.16 Carbohydrate Metabolism:
Other Important Sugars
Section 8.5: Glycogen Metabolism
Glycogenesis
Synthesis of glycogen, the storage form of glucose,
occurs after a meal
Requires a set of three reactions (1 and 2 are
preparatory and 3 is for chain elongation):
1. Synthesis of glucose-1-phosphate (G1P) from glucose6-phosphate by phosphoglucomutase
2. Synthesis of UDP-glucose from G1P by UDP-glucose
phosphorylase
Section 8.5: Glycogen Metabolism
Figure 8.17a Glycogen
Synthesis
Glycogen
synthase
Glycogenesis Continued
3. Synthesis of Glycogen from UDP-glucose requires two
enzymes:
 Glycogen synthase to grow the chain
Section 8.5: Glycogen Metabolism
Glycogenesis
Continued
Branching enzyme
amylo-a(1,41,6)glucosyl transferase
creates a(1,6)
linkages for
branches
Branching
enzyme
a(1,6) Glycosidic Linkage is formed
Figure 8.17b Glycogen Synthesis
Section 8.5: Glycogen Metabolism
Glycogenolysis
Glycogen degradation requires two reactions:
1. Removal of glucose from nonreducing ends (glycogen
phosphorylase) within four glucose of a branch point
Section 8.5: Glycogen Metabolism
Figure 8.18 Glycogen
Degradation
Section 8.5: Glycogen Metabolism
Glycogenolysis Cont.
Glycogen degradation
requires two reactions:
2. Hydrolysis of the
a(1,6) glycosidic
bonds at branch
points by amyloa(1,6)-glucosidase
(debranching
enzyme)
Amylo-a(1,6)-glucosidase
Amylo-a(1,6)-glucosidase
Figure 8.19 Glycogen Degradation
via Debranching Enzyme
Section 8.5: Glycogen Metabolism
Amylo-a(1,6)-glucosidase
Figure 8.19 Glycogen Degradation via Debranching Enzyme
Section 8.5: Glycogen Metabolism
Regulation of
Glycogen Metabolism
Figure 8.21 Major Factors Affecting
Glycogen Metabolism
Carefully regulated
to maintain
consistent energy
levels
Regulation involves
insulin, glucagon,
epinephrine, and
allosteric effectors
Section 8.5: Glycogen Metabolism
Glucagon activates
glycogenolysis
Insulin inhibits
glycogenolysis and
activates glycogenesis
Epinephrine release
activates
glycogenolysis and
inhibits glycogenesis
Figure 8.21 Major Factors Affecting
Glycogen Metabolism