Section 3 Proteins, Enzymes and Central Metabolism.
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Transcript of Section 3 Proteins, Enzymes and Central Metabolism.
Section 3
Proteins, Enzymes and Central Metabolism
Chapter 5
Amino Acids, Peptides, & Proteins
Section 5.1: Amino Acids
Proteins are molecular toolsThey are a diverse and complex group of macromolecules
Figure 5.1 Protein Diversity
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 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
Figure 5.3 General Structure of the a-Amino Acids
Section 5.1: Amino Acids
Amino Acid Classes Classified by their ability to interact with water Nonpolar amino acids contain hydrocarbon
groups with no charge
Figure 5.2 The Standard Amino Acids
Section 5.1: 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)
Figure 5.2 The Standard Amino Acids
Section 5.1: Amino Acids
Amino Acid Classes ContinuedAcidic 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
Figure 5.2 The Standard Amino Acids
Section 5.1: Amino Acids
Section 5.1: Amino Acids
Biologically Active Amino Acids Amino acids can have other
biological roles1. Some amino acids or derivatives can act as chemical messengers
Neurotransmitters (tryptophan- derivative 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
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)
Figure 5.7 Modified Amino Acid Residues Found in Polypeptides
Section 5.1: Amino AcidsAmino 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
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
Figure 5.9 D- and L-Glyceraldehyde
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 + pK2pI = 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
Amino acids with ionizable side chains have more complex titration curves
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 a-carboxyl group loses a proton
As more base is added, it loses protons to a final net charge of -2
Figure 5.10 Titration of Two Amino Acids: Glutamic Acid
+10
-1
-2
pK1+pKR= pKI
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 double-bond 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 possibilitiesFigure 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
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
Figure 5.16 Segments of b-chain in HbA and HbS
Section 5.3: Proteins
Figure 5.18 The a-Helix
Secondary Structure: Polypeptide secondary structure has a variety of repeating structures
Most common include the a-helix and b-pleated 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
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
Figure 5.20 Selected Supersecondary Structures
Section 5.3: Proteins Tertiary Structure refers to unique three-
dimensional 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 features1. Many polypeptides fold in a way to bring distant amino acids into close proximity2. 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 proximity2. Globular proteins are compact because of efficient packing3. 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
Interactions that stabilize tertiary structure are hydrophobic interactions, electrostatic interactions (salt bridges), hydrogen bonds, covalent bonds, and hydration
Figure 5.23 Interactions That Maintain Tertiary Structure
Section 5.3: Proteins
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)
Figure 5.25 Structure of Immunoglobulin G
Section 5.3: Proteins
Reasons for common occurrence of multisubunit proteins:
1. Synthesis of subunits may be more efficient2. In supramolecular complexes replacement of worn-out components can be handled more effectively 3. Biological function may be regulated by complex interactions of multiple subunitsFigure 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
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)
Figure 5.27 Disordered Protein Binding
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
Figure 5.28 The Anfinsen Experiment
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 site-directed 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
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 d- chains instead of b-chains (HbA2)
Embryonic and fetal hemoglobin have e- and g-chains that have a higher affinity for O2
Figure 5.38 The Oxygen-Binding Site of Heme Created by a Folded Globin Chain
Section 5.3: Proteins
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
Figure 5.39 Hemoglobin
Section 5.3: Proteins
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
Figure 5.41 Equilibrium Curves Measure the Affinity of Hemoglobin and Myoglobin for Oxygen
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
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
Figure 5.42 The Effect of 2,3-Bisphosphoglycerate (BPG) on the Affinity Between Oxygen and Hemoglobin
Section 5.3: Proteins
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)
Section 5.4: Molecular Machines
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
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
Figure 6.1 A Catalyst Reduces the Activation Energy of a Reaction
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
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 holoenzymesThe protein component is the apoenzyme
Figure 6.2 The Induced Fit Model
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. Oxidoreductases2. Transferases3. Hydrolases4. Lyases5. Isomerases6. 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)
Half-life is the time for one-half of the reactant molecules to be consumed
Figure 6.3b Enzyme Kinetic Studies
Rate = k[A]1
Section 6.3: Enzyme Kinetics
Second order is bimolecular (A + B P)
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
Rate = k[A]1[B]1
Section 6.3: Enzyme Kinetics
Michaelis-Menten Kinetics The concept of enzyme substrate complexes:
Introduce the Michaelis constant Km
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
k1E + S ES E + P k-1
k2
Km = k-1 + k2
k1
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
The specificity constant reflects the relationship between catalytic rate and substrate binding affinity (kcat/Km)
Specific activity is a measure used to identify enzyme purificationFigure 6.5 A Michaelis-
Menten Plot
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
Slope of the line Km/Vmax
1/Vmax is the Y intercept
-1/Km is the X interceptFigure 6.6 Lineweaver-Burk or Double-Reciprocal Plot
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
Forms enzyme-inhibitor (EI) complex
Increasing substrate concentration overcomes competitive inhibition
Figure 6.8 Michaelis-Menten Plot of Uninhibited Enzyme Activity Versus Competitive Inhibition
Section 6.3: Enzyme Kinetics
Noncompetitive Inhibitors can bind reversibly to the ES complex at a site other than the active site
Forms EI + S and EIS complex
Changes enzyme conformation
Increased substrate concentration partially reverses inhibition
This is the case for pure noncompetitive inhibition only
Figure 6.10 Michaelis-Menten Plot of Uninhibited Enzyme Activity Versus Noncompetitive Inhibition
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:
double-reciprocal plots may be used to distinguish competitive, noncompetitive, and uncompetitive inhibition
Section 6.3: Enzyme Kinetics
Competitive inhibition increases Km, not Vmax (6.10a)
Pure noncompetitive Vmax lowered Km unchanged (6.10b)
Figure 6.11 Kinetic Analysis of Enzyme Inhibition
Section 6.3: Enzyme Kinetics
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)
Figure 6.11 Kinetic Analysis of Enzyme Inhibition
Section 6.3: Enzyme Kinetics
Uncompetitive—Km and Vmax are changed although ratio is the same (6.10e)
Figure 6.11 Kinetic Analysis of Enzyme Inhibition
Section 6.3: Enzyme Kinetics
Allosteric Enzymes have a sigmoidal curve rather than a hyperbolic one
Resembles the oxygen-binding curve of hemoglobin
Michaelis-Menten kinetics do not apply to allosteric enzymes
Figure 6.13 The Kinetic Profile of an Allosteric Enzyme
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
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 electron-rich 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
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
Section 6.4: Catalysis
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
Figure 6.14 Energy Profile for a Two-Step Reaction
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
Section 6.4: 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
Figure 6.15 Ester Hydrolysis: Hydroxide Ion Catalysis
Section 6.4: 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
Figure 6.15 Ester Hydrolysis: General Base Catalysis
Section 6.4: Catalysis
Covalent Catalysis—the formation of an unstable covalent bond with a nucleophilic group on the enzyme and an electrophilic group on the substrate
Figure 6.15 Ester Hydrolysis: General Acid 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
Enzymes are proteins and become denatured at high temperatures
Section 6.4: Catalysis
Figure 6.16 The Effect of Temperature on Enzyme Activity
Section 6.4: Catalysis
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
Figure 6.17 The Effect of pH on Two Enzymes
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
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
Alcohol Dehydrogenase—catalyzes the reversible oxidation of alcohols to aldehydes or ketones
Uses NAD+ as a hydride (H:-) ion acceptor
Figure 6.19 Alcohol Dehydrogenase
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
Section 6.5: Enzyme Regulation
Figure 6.20 The Activation of Chymotrypsinogen
Covalent Modification 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 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
Figure 6.21 The Rate of an Enzyme-Catalyzed Reaction as a Function of Substrate Concentration
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 6.22a 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 6.22b 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
Figure 6.22 Allosteric Interaction Models
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
Section 6.5: Enzyme Regulation
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
Energy transforming pathways of carbohydrate metabolism include glycolysis, glycogenesis, glycogenolysis, gluconeogenesis, and pentose phosphate pathway
Figure 8.2 Major Pathways in Carbohydrate Metabolism
Section 8.1: Glycolysis
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
Figure 8.2 Major Pathways in Carbohydrate Metabolism
Section 8.1: Glycolysis
Glycolysis is an anaerobic processTwo 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 H2O
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-6-phosphate
Phosphorylation of glucose (kinase) prevents transport out of the cell and increases reactivity
2. Conversion of glucose-6-phosphate to fructose-6-phosphate
Conversion of aldose to ketose
Figure 8.3a Glycolytic Pathway
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-1,6-bisphosphate
Aldol cleavage giving an aldose and ketose product
Figure 8.3a Glycolytic Pathway
Section 8.1: Glycolysis
Reactions of the Glycolytic Pathway Continued
5. Interconversion of glyceraldehyde-3-phosphate 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 duplicate6. Oxidation of glyceraldehyde- 3-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
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
Figure 8.4Glyceraldehyde-3-Phosphate Dehydrogenase Reaction
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 formFigure 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
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
Figure 8.6 The Fates of Pyruvate
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
Energetics of Glycolysis In red blood cells, only three reactions have significantly negative DG values
Figure 8.8 Free Energy Changes during Glycolysis in Red Blood Cells
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 hormone-
induced covalent modification of PFK-2, activates PFK-1
Accumulation of fructose-1,6-bisphosphate activates PFK-1 providing a feed-forward mechanism
Figure 8.9 Fructose-2,6-Bisphosphate Level Regulation
Section 8.1: Glycolysis
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 acidsGluconeogenesis Reactions
Reverse of glycolysis except the three irreversible reactions
Section 8.2: Gluconeogenesis
Figure 8.10 Carbohydrate Metabolism: Gluconeogenesis and Glycolysis
Section 8.2: GluconeogenesisFigure 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 carboxykinase2. Conversion of fructose-1,6-bisphosphate to fructose-6-phosphate via the enzyme fructose-1,6-bisphosphatase3. Formation of glucose from glucose-6-phosphate via the liver and kidney-specific enzyme glucose-6-phosphatase
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
Gluconeogenesis Substrates Continued 3. Alanine—generated from pyruvate in
exercising muscle Alanine is converted to pyruvate and then
glucose in the liver
Figure 8.12 The Glucose Alanine Cycle
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-1,6-bisphosphatase, and glucose-6-phosphatase)
Figure 8.13 Allosteric Regulation of Glycolysis and Gluconeogenesis
Section 8.3: Pentose Phosphate Pathway
Pentose Phosphate Pathway Alternate glucose
metabolic pathway Products are NADPH
and ribose-5-phosphate
Two phases: oxidative and nonoxidative
Figure 8.14a The Pentose Phosphate Pathway (oxidative)
Glucose-6-phosphate dehydrogenase
Gluconolactonase
Pentose Phosphate Pathway: Oxidative Three reactions Results in ribulose-
5-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
6-phosphogluconate dehydrogenase
Pentose Phosphate Pathway: Nonoxidative Produces important
intermediates for nucleotide biosynthesis and glycolysis
Ribose-5-phosphate Glyceraldehyde-3-
phosphate 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 8.3: Pentose Phosphate Pathway
Section 8.4: Metabolism of Other Important Sugars
Fructose, mannose, and galactose are also important sugars for vertebrates Most common sugars found in oligosaccharides
besides glucose
Figure 8.16 Carbohydrate Metabolism: Galactose Metabolism
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
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 glucose-6-phosphate by phosphoglucomutase2. Synthesis of UDP-glucose from G1P by UDP-glucose phosphorylase
Section 8.5: Glycogen Metabolism
Glycogenesis Continued3. Synthesis of Glycogen from UDP-glucose requires two enzymes:
Glycogen synthase to grow the chain
Figure 8.17a Glycogen Synthesis
Section 8.5: Glycogen Metabolism
Glycogen synthase
Branching enzyme
Section 8.5: Glycogen Metabolism
Glycogenesis Continued
Branching enzyme amylo-a(1,41,6)-glucosyl transferase creates a(1,6) linkages for branches
Figure 8.17b Glycogen Synthesis
a(1,6) Glycosidic Linkage is formed
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
Section 8.5: Glycogen Metabolism
Figure 8.19 Glycogen Degradation via Debranching Enzyme
Glycogenolysis Cont. Glycogen degradation
requires two reactions:
2. Hydrolysis of the a(1,6) glycosidic bonds at branch points by amylo-a(1,6)-glucosidase (debranching enzyme)
Amylo-a(1,6)-glucosidase
Amylo-a(1,6)-glucosidase
Section 8.5: Glycogen Metabolism
Figure 8.19 Glycogen Degradation via Debranching Enzyme
Amylo-a(1,6)-glucosidase
Regulation of Glycogen Metabolism Carefully regulated
to maintain consistent energy levels
Regulation involves insulin, glucagon, epinephrine, and allosteric effectors
Section 8.5: Glycogen Metabolism
Figure 8.21 Major Factors Affecting Glycogen Metabolism
Figure 8.21 Major Factors Affecting Glycogen Metabolism
Section 8.5: Glycogen Metabolism
Glucagon activates glycogenolysis
Insulin inhibits glycogenolysis and activates glycogenesis
Epinephrine release activates glycogenolysis and inhibits glycogenesis