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Transcript of Biochemical Engineering Lecture
8/12/2019 Biochemical Engineering Lecture
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Biochemical Engineering
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BASICMICROBIOLOGY
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Protist Kingdom
Bacteria Blue green
algae
Fungi Algae Protozoa
Molds YeastEubacteria Archaeobacteria
Prokaryotes Eucaryotes
CLASSES OF ORGANISMS
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PROKARYOTIC CELLS
They do not have a nucleus
They have no membrane-boundorganelles
The parts are – Cell wall
– Plasma membrane
– Ribosomes – Flagella
– Pili
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The Bacterial Cell
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EUKARYOTIC CELLS
Plant Cell
Animal Cell
Parts – Nucleus
– Plasma membrane – Organelles Endoplasmic recticulum
– Rough
– Smooth
Golgi Complex Mitochondrion
Lysosome
Chloroplast
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An Animal Cell
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A Plant Cell
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THE NUCLEUS It houses the chromatin, which is a mass of
DNA and protein. During cell division the chromatin coils up into
recognizable chromosomes.
The nuclear envelope is a double membraneperforated with pores that allow transport ofmaterials back and forth to the cyotplasm.
The nucleus is the site of DNA replication andRNA synthesis (transcription). It is the site ofthe control of gene expression.
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ROUGH ENDOPLASMIC RECTICULUM
It is rough because imbedded in the membrane areribosomes.
It is the site of the synthesis of secretory proteins.
It is the site for the synthesis of membrane. Enzymes
synthesize phospholipid that forms all themembranes of the cell.
Ribosomes in the rough ER synthesize protein thatare then converted to glycoprotein and packaged in
transport vesicles for secretion.
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SMOOTH ENDOPLASMIC RECTICULUM
The smooth ER is the site for the synthesis oflipids, phospholipids, and steroids.
The production of steriod hormones is tissue
specific. – For example, it is the smooth ER of the cells of the
ovaries and testes that synthesize the sexhormones.
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The smooth ER of the liver has several additionalfunctions. Enzymes in the smooth ER regulate the
release of sugar into the bloodstream while otherenzymes break down toxic chemicals. As the liver isexposed to additional doses of a drug the liverincreases the amount of smooth ER to handle it. It
then takes more drug to get past the detoxifiyingability of the liver. We become more tolerant of thedrug.
Finally the smooth ER functions to store calcium ions.Ca+ ions are required for muscle contraction.
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THE GOLGI COMPLEX
The Golgi apparatus, like the ER, is a seriesof folded membranes.
It functions in processing enzymes and other
products of the ER to a finished product.
It is the source of the production oflysosomes
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MITOCHONDRIA
These organelles are the sites of respirationand convert the chemical energy of sugarsand other organic compounds into the high-energy phosphate bonds of an ATP molecule.
These are also bound by a doublemembrane. The inner membrane is thefolded (the folds are called cristae) and is the
site of the electron transport system.
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LYSOSOMES
These are membrane bound vesicles that
harbor digestive enzymes.
The membrane of a lysosome will fuse with
the membrane of vacuoles and releases thesedigestive enzymes to the interior of thevacuole to digest the material inside thevacuole.
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VACUOLES
These are membrane bound sacs that have
many different functions.
– The central vacuole of a plant cell serves as alarge lysosome.
– It may also function in absorbing water.
– The central vacuoles of flower petal cells may holdthe pigments that give the flower its color.
– The contractile vacuoles of protists collect and
excrete water.
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Lysosome Formation and Function
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CILIA AND FLAGELLA These are found on cells, such as protists, that are
motile.
Cilia are short and numerous.
Flagella are longer and less numerous appendages
These are composed of a core of microtubules wrappedin an extension of the plasma membrane.
It is sufficient to know that Energy is required to movethe cilia or flagella in a whip-like motion to propel thecell.
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PROKARYOTIC CELLS EUCARYOTIC CELLS
1 – 5 m in length 10 – 30 m in length
Genetic material is a nucleoid, a poorlydemarcated region of the cell that lacks aboundary membrane to separate it fromsurrounding cytoplasm.
Possess a nucleus, a region bounded by acomplex membranous structure callednuclear envelop.
Contain relatively small amount of DNA
Length of DNA ranges from 0.25mm to3mm.
Single chromosome consists of essentiallynaked DNA
Contain several orders of magnitude ofmore genetic information
Length of DNA is about 4.6mm (yeast)
Chromosome consists of fibers containingboth DNA and protein
Cytoplasm is essentially devoid ofmembranous structure
Cytoplasm is filled with a great diversity ofstructures
No condensation of chromosomes and no
spindle apparatus. The DNA is duplicatedand the two copies are simply separatedby the growth of an intervening cellmembrane
Divide by a complex process of mitosis in
which duplicated chromosomes condenseinto compact structures that are separatedby an elaborate microtubule-containingapparatus
Simple locomotion mechanism Possess complex loco motor mechanism
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The Chemistry of Life
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The elements of life:
Stars (with assistance from the Big bang) have formed83 stable chemical elements in the universe
~95% of the mass of all terrestrial organisms composedof just 4 of them
– Hydrogen (61% in humans)
– Oxygen (26% in humans)
– Carbon (10.5% in humans) – Nitrogen (2.4% in humans)
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CARBOHYDRATES
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Polysaccharides
Polymers composed of sugars /saccharides
Uses include energy source, component ofextra cellular matrix
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Monosaccharide / Disaccharide
DISACCHARIDES
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DISACCHARIDES
MALTOSE
Lactose = -D-galactose + glucose
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What is Starch?
The term starch is used to describe a biopolymer systemcomprising predominantly of two polysaccharides - amylose
and amylopectin.
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AmyloseThe smaller of the two polysaccharides which make up starch, amylose is a
linear molecule comprising of (1-4) linked alpha-D-glucopyranosyl units.
Figure 2 : Amylose molecule
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Amylopectin
The larger of the two components, amylopectin is highly branched with a much
greater molecular weight. This structure contains alpha-D-glucopyranosyl units
linked mainly by (1-4) linkages (as amylose) but with a greater proportion of (1-
6) linkages, which gives a large highly branched structure.
Amylopectin has been found to form the basis of the structure of starch
granules. This is because the short branched (1-4) chains are able to form
helical structures which crystallise.
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Cellulose : the major structural component of woody
plants and natural fibers such as cotton, wood, and cork, is a
ß-D-glucose polymer found in vegetable matter.
The ß-glycoside linkages in cellulose give the glucose rings a different
relative orientation than is found in starch. Although this difference may
seem minor, it has very important consequences : human being are not able
to digest them
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LIPIDS
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Lipids and Phospholipids
Long hydrocarbon chains with activegroup on one end
– Fatty acids
– Neutral fats
– Phospholipids (fatty acid derivatives found incell membranes)
Structure formation is analogous tosurfactant, block copolymer
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Lipid Classessimple: FA‟s esterified with glycerol compound: same as simple, but with other compoundsalso attached
phospholipids: fats containing phosphoric acid andnitrogen (lecithin)
glycolipids: FA‟s compounded with CHO, but no N
derived lipids: substances from the above derived by
hydrolysis
sterols: large molecular wt. alcohols found in nature andcombined w/FA‟s (e.g., cholesterol)
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Nutritional Uses of Lipids
We already know that lipids are concentratedsources of energy (9.45 kcal/g)
other functions include:
1) provide means whereby fat-soluble nutrients(e.g., sterols, vitamins) can be absorbed by the body
2) structural element of cell, subcellular components
3) components of hormones and precursors forprostaglandin synthesis
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Omega fatty acids
Polyunsaturated fatty acids like DHA and EPA are added to
many foods to due to their nutritive value. They are present
naturally to the highest levels in fish oils.
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Triglycerides
A FAT MOLECULE = GLYCEROL + FATTY ACID
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A FAT MOLECULE GLYCEROL + FATTY ACID
The three fatty acids in a single fat molecule may be all alike (as
shown here for tristearin) or they may be different. They may
contain as few as 4 carbon atoms or as many as 24.
E ti l F tt A id
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Essential Fatty AcidsNeeded by body but cannot be synthesized so
external source required
LINOLEIC CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH
18:2 n-6LINOLENIC CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH
18:3 n-3
EICOSOPENTAENOIC ACID
CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COO
H
20:5 n-3
DOCOSOHEXAENOIC ACID 22: 6 n-3
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PROTEINS
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Proteins
• Proteins are polymers composed of amino acid
monomers
• Polypeptides is another term for amino acid polymers
• Proteins are characterized by a specific primary
structure – order of mers in the backbone and DP
• Control of primary structure leads to control of 3Dstructure
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Proteins
The control of protein structure buildsinformation into the molecule that translatesinto function
Proteins are the most common biological
macromolecules in the extra cellular matrix Perform structural and functional tasks
– Collagen (triple helix – gly-X-Y) where proline andhydroxy proline is often present is the basicstuctural protein
– Enzymes perform specific catalytic tasks
– Adhesive proteins are bind cells to substrates – fibronectin, integrin, etc.
– Provide signal transduction between cells and ECM
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Peptide Synthesis
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Protein Structure Hierarchy
Secondary structure refers to local chainconformations – four types are known:
– helix – regular helix
– sheet – extended zig-zag – turn – puts fold into sheet
– Globular or random coil
Tertiary structure refers to secondary structure
stabilized by H bonds – defines protein folding
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NUCLEIC ACIDS
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DNA Chemistry
DNA is a complex molecule which is built ofthree basic types of monomers:
– 1. Sugar (deoxyribose)
– 2. A phosphate PO4 – 3. One of four “nitrogenous bases”
Adenine (A)
Guanine (G)
Cytosine (C) Thymine (T)
– These four monomers are collectively called“nucleotides”
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The DNA Nitrogenous Bases:
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Differences between
DNA and RNA
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DNA construction
The double helix:
– Resembles a twisted ladder
The “rails” of the DNA ladder are made of the
sugar and phosphate
The “rungs” of the ladder are composed of one offour pairs of the nitrogenous bases
– Either AT, TA, GC or CG
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DNA Letters, Genes
The rungs of the double helix are like the mapon the floor. They spell out which amino acidshould line up where – Each rung can have one of four possible “letters”
AT
TA
GC
CG
– Each slot where an amino acid will line up is formedof three rungs of the double helix A set of three rungs is called a “gene”
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DNA and amino acids
Each gene (three rungs) matches upchemically to one of the 20 aminoacids used by life• Each gene „spells‟ the name of an amino acid! • The amino acids line up along the double
helix according to the map spelled out by thesequences of sets of three rungs
• They the amino acid monomers join together
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The Nucleic Acid
Complex structures used tomaintain genetic information
DNA – deoxyribonucleic acidserves as the “Master Copy” formost information in the cell.
RNA – Ribonucleic acid acts totransfer information from DNA tothe rest of the cell.
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The RNA Phosphoric acid
Ribose ( a pentose)
Organic (nitrogeneous) bases:
– Purines: Adenine & guanine
– Pyrimidines: Cytosine and Uracil
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Th DNA
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The DNA
DNA is a polymer known as
polynucleotide
• Each nucleotide consist of
-5 carbon sugar
-Nitrogen containing baseattached to the sugar
• There are 4 nucleotides
- Adenine- Guanine
- Thymine
- Cytosine
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Translation of RNA to Protein
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Translation of RNA to Protein
I iti ti
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Initiation
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Elongation
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Termination
U C
A G
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U UUU = Phe
UUC = Phe
UUA = Leu
UUG = Leu
UCU = Ser
UCC = Ser
UCA = Ser
UCG = Ser
UAU = Tyr
UAC = Tyr
UAA = Stop
UAG = Stop
UGU = Cys
UGC = Cys
UGA = Stop
UGG = Trp U
C
A
G
C CUU = Leu
CUC = Leu
CUA = Leu
CUG = Leu
CCU = Pro
CCC = Pro
CCA = Pro
CCG = Pro
CAU = His
CAC = His
CAA = Gln
CAG = Gln
CGU = Arg
CGC = Arg
CGA = Arg
CGG = Arg U
C
A
G
A AUU = Ile
AUC = Ile
AUA = Ile
AUG = Met
ACU = Thr
ACC = Thr
ACA = Thr
ACG = Thr
AAU = Asn
AAC = Asn
AAA = Lys
AAG = Lys
AGU = Ser
AGC = Ser
AGA = Arg
AGG = Arg U
C
A
G
G GUU = ValCUC = Val
GUA = Val
GUG = Val GCU = AlaGCC = Ala
GCA = Ala
GCG = Ala GAU = AspGAC = Asp
GAA = Glu
GAG = Glu GGU = GlyGCG = Gly
GGA = Gly
GGG = Gly U
C
A
G
AUG = start codon
UAA, UAG, and UGA = stop (nonsense) codons
Amino Acids
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Amino Acids
Phe = phenylalanine
Leu = leucine
Ile = isoleucine
Met = methionine
Val = valine
Ser = serine
Pro = proline
Thr = threonine
Ala = alanine
Tyr = tyrosine
His = histidineGln = glutamine
Asn = asparagine
Lys = lysine
Asp = aspartic acid
Glu = glutamic acid
Cys = cysteine
Trp = tryptophanArg = arginine
Gly = glycine
S t M t ti
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Spontaneous Mutation
•
Substitution of a nucleotide (point mutations)
Spontaneous Mutation
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•Deletion or addition of a nucleotide
Spontaneous Mutation
Results of Spontaneous Mutation
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Results of Spontaneous Mutation
Missense mutation This is usually seenwith a single substitution mutation and
results in one wrong codon and one
wrong amino acid
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Nonsense mutation - If the change in the
deoxyribonucleotide base sequence
results in transcription of a stop ornonsense codon, the protein would be
terminated at that point in the message
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Sense mutation - This is sometimes
seen with a single substitution
mutation when the change in the
DNA base sequence results in a new
codon still coding for the same
amino acid.
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Frameshift Mutation - This is seen when a
number of DNA nucleotides not divisible
by three is added or deleted.
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ENZYME KINETICS AND APPLICATIONS
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Enzymes
• Enzymes endow cells with the remarkable capacity
to exert kinetic control over thermodynamic
potentiality
• Enzymes are the agents of metabolic function
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ENZYMES ARE NAMED BY WHAT THEY DO RATHER THAN WHAT THEY
ARE. NAME ENDS WITH …-ASE. EG. AMYLASE
E Bi l i l
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– Enzymes as BiologicalCatalysts
Increase reaction ratesby over 1,000,000-fold
Two fundamentalproperties
– Increase the reaction ratewith no alteration of theenzyme
– Increase the reaction ratewithout altering the
equilibrium Reduce the activation
energy
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– Enzymes as Biological Catalysts
The substrate binds to a specific region called theactive site
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– Enzymes as Biological
Catalysts Two popular models
provide an aid tounderstanding the
mechanisms of enzymeaction:
– Lock-and-key
– Induced fit
The M ichaelis Menten Equation
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The M ichaelis-Menten Equation
Louis Michaelis and Maude Menten’s theory
It assumes the formation of an enzyme-substrate
complex
It assumes that the ES complex is in rapid equilibriumwith free enzyme
Breakdown of ES to form products is assumed to be
slower than 1) formation of ES and 2) breakdown of ES
to re-form E and S
Plot initial velocity against substrate concentration
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V
[S]
(Vmax)
E + S ES E + Pk 1 k 2
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E + S ES E + Pk -1 k -2
The following assumptions allow Michaelis-Menten model to
explain V vs S kinetics
1. Enzyme and substrate combine to form ES complex
2. Assume reverse rxn, k-2, is negligible
3. Assume [ES] is constant, steady state assumption: d[ES]/dt = 0
4. [E] <<<[S]
E + S ES E + Pk 1 k 2
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E + S ES E + Pk -1
[Et] [S][ES] = -------------------------
[S] + (K2+ K-1) / K1
Define Km as (K2+ K-1) / K1
(as the Michaelis-Menten constant)
[Et] [S]
[ES] = -----------------
[S] + km
E + S ES E + Pk 1 k 2
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E + S ES E + Pk -1
V0 = K2 [ES]K2 [Et] [S]
= ------------------
[S] + km
Maximum velocity (Vmax) occurs when [ES] = [Et]
Thus, Vmax = K2 [Et]
Vmax [S]
V0 = ------------------ (Michaelis-Menten equation)
km + [S]
E + S ES E + Pk 1 k 2
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E + S ES E + Pk -1
Vmax [S]V0 = ------------------
km + [S]
When V0 = 1/2 Vmax
Vmax Vmax [S]
--------------- = ------------------
2 km + [S]
Solve for km
Km = [S] when V0 = 1/2 Vmax
Let's find Vmax & Km on the graph
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Vmax = v at highest [S]
Km = [S] at 1/2 Vmax
Understanding K
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Understanding K m
The “kinetic activator constant”
K m is a constant
K m is constant derived from rate constants
K m is, under true Michaelis-Meten conditions,estimate of the dissociation constant of E from S
Small K m means tight binding; high K m means weak
binding
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Understanding V max
The theoretical maximal velocityVmax is a constant
Vmax is the theoretical maximal rate of the reaction –
but it is NEVER achieved in reality
To reach Vmax would require that all enzyme
molecules are tightly bound with subtrate
Vmax is asymptotically approached as substate is
increased
The dual nature of the M ichaelis-
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The dual nature of the M ichaelis
Menten equation
Combination of 0-order and 1 st -order kinetics
When S is low, the equation for rate is 1st-order in S
When S is high, the equation for rate is 0-order in SThe Michaelis-Menten equation diescribes a
rectangular hyperbolic dependence of v on S!
Th b
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The turnover number
A measure of catalytic activity
k cat, the turnover number, is the number of
substrate molecules converted to product per
enzyme molecule per unit of time, when E issaturated with substrate
If the M-M model fits, k 2 = k cat = Vmax/Et
Values of k cat
range from less than 1/sec to many
millions per sec
Vmax [S]
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Vmax [S]
V0 = ------------------
km + [S]
1 km + [S]
--------- = ------------------
V0 Vmax [S]
1 km [S]---------- = ------------ + ------------
V0 Vmax [S] Vmax [S]
1 km 1 1---------- = ------ ------ + ------
V0 Vmax [S] Vmax
( This is the Lineweaver-Burk equation)
Lineweaver-Burk Plot: 1/V0 against 1/[S]
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0 g [ ]
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Enzyme I nhibitors
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Enzyme I nhibitors
Competitive vs. Noncompetitive
SvCompetitive Inhibition
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I
M K I S K
S v v
max
Competitive Inhibition
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Non-Competitive Inhibition