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


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


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


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)



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



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


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


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


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


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!


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


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


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


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


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


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)


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)


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


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


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


Figure 5.19 b-Pleated Sheet

The b-pleated sheets form when two or more polypeptide chain segments line up, side by side


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


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


Figure 5.21 Selected Domains Found in Large Numbers of 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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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)


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


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


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


Competitive inhibition increases Km, not Vmax (6.10a)

Pure noncompetitive Vmax lowered Km unchanged (6.10b)

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)



Uncompetitive—Km and Vmax are changed although ratio is the same (6.10e)



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


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


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

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



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


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


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


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


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)


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


Figure 6.16 The Effect of Temperature on Enzyme Activity


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



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


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



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


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


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


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


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


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


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


Figure 8.3 Glycolytic Pathway


Figure 8.3 Glycolytic

Pathway


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


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




5. Interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate

Conversion of aldose to ketose enables all carbons to continue through 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)


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


Figure 8.4 Glyceraldehyde-3-Phosphate Dehydrogenase Reaction



7. Phosphoryl group transfer

Production of ATP via substrate-level phosphorylation




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)



10. Synthesis of pyruvate Formation of pyruvate and ATP

Produces a net of 2 ATP, 2 NADH, and 2 pyruvate



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


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


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


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


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


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 8.2: 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


Figure 8.10 Carbohydrate Metabolism: Gluconeogenesis and Glycolysis

Section 8.2: GluconeogenesisFigure 8.10 Carbohydrate Metabolism:

Gluconeogenesis and Glycolysis


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


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


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


Gluconeogenesis Regulation Substrate availability Hormones (e.g.,

cortisol and insulin)

Figure 8.13 Allosteric Regulation of Glycolysis and 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)


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)


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

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


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)


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


Glycogen synthase

Branching enzyme


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


Figure 8.18 Glycogen Degradation



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



Figure 8.19 Glycogen Degradation via Debranching Enzyme


Regulation of Glycogen Metabolism Carefully regulated

to maintain consistent energy levels

Regulation involves insulin, glucagon, epinephrine, and allosteric effectors


Figure 8.21 Major Factors Affecting Glycogen Metabolism

Figure 8.21 Major Factors Affecting Glycogen Metabolism


Glucagon activates glycogenolysis

Insulin inhibits glycogenolysis and activates glycogenesis

Epinephrine release activates glycogenolysis and inhibits glycogenesis

section 3 proteins, enzymes and central metabolism

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