BIOCHEM REVIEW

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BIOCHEM REVIEW 1

• Jason Emer
• jemer1_at_uic.edu
• Obi Ekwenna
• oekwen1_at_uic.edu

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YOUR TEST

• Monday, September 13
• 2-3 questions per Lec. (26-39)
• Pass Level 55-65

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Acid-Base Chemistry

• Acid a proton donor
• Base accepts protons
• HA lt-----gt A- H
• Ka HA-/HA
• pKa -logKa

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Acid-Base Chemistry

• Weak acids and bases in solution do not fully
  dissociate and, therefore, there is an
  equilibrium between the acid and its conjugate
  base.
• This equilibrium can be calculated and is termed
  the equilibrium constant Ka. This is also
  sometimes referred to as the dissociation
  constant as it pertains to the dissociation of
  protons from acids and bases.

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Acid-Base Chemistry

• Why is this important?
• pKa pH -logA-/HA
• By rearranging the above equation we arrive at
  the Henderson-Hasselbalch equation
• pH pKa logA-/HA

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Acid-Base Chemistry

• Clinical Significance.Blood Buffering
• The pH of blood is maintained in a narrow range
  around 7.4.
• Even relatively small changes in this value of
  blood pH can lead to severe metabolic
  consequences.
• Therefore, blood buffering is extremely important
  in order to maintain homeostasisAka Ying
  Yang..not the Twins.

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Acid-Base Chemistry

• The primary buffers in blood are hemoglobin in
  erythrocytes and bicarbonate ion (HCO3-) in the
  plasma.
• The formation of bicarbonate ion in blood from
  CO2 and H2O allows the transfer of relatively
  insoluble CO2 from the tissues to the lungs,
  where it is expelled. The major source of CO2 in
  the tissues comes from the oxidation of ingested
  carbon compounds

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Acid-Base Chemistry
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Acid-Base Chemistry

• CO2 H2O lt------gt H2CO3
• H2CO3 lt-------gt H HCO3-

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Acid-Base Chemistry

• If blood is not adequately buffered, the result
  may be metabolic acidosis or metabolic alkalosis.
  
• These physiological states can be reached if a
  metabolic defect results in the inappropriate
  accumulation or loss of acidic or basic
  compounds.
• These compounds may be ingested, or they may
  accumulate as metabolic by-products such as
  acetoacetic acid and lactic acid. Both of these
  will ionize, thereby increasing the level of H
  ions that will in turn remove bicarbonate ions
  from the blood and alter blood pH. The
  predominant defect in acid or base elimination
  arises when the excretory system of the kidneys
  is impaired.
• Alternatively, if the lungs fail to expel
  accumulated CO2 adequately and CO2 accumulates in
  the body, the result will be respiratory
  acidosis. If a decrease in PCO2 within the lungs
  occurs, as during hyperventilation, the result
  will be respiratory alkalosis.

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Acid-Base Chemistry

• Typical Questions
• What is the pKa of an acid which has a pH of 6.8
  when its baseacid ratio is 120? Dang!!! We ve
  to know that pH eqn! YES!
• A patient comes into the emergency room feeling
  faint. He says that he is an insulin-dependent
  diabetic. His blood gases have been determined
  and you see his value for HCO3- 18
  miliequivalents per liter and for PCO2, 38
  mmHg. Based on your calculations this patient
  has? Acidosis? Alkalosis? Danger or Not?

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Amino Acids, Polypeptides Proteins

• All peptides and polypeptides are polymers of
  alpha-amino acids.
• There are 20 ?-amino acids that are relevant to
  the make-up of mammalian proteins.
• Several other amino acids are found in the body
  free or in combined states (i.e. not associated
  with peptides or proteins).

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Amino Acids, Polypeptides Proteins

• The ?-amino acids in peptides and proteins
  (excluding proline) consist of a carboxylic acid
  (-COOH) and an amino (-NH2) functional group
  attached to the ?- carbon atom.

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Amino Acids, Polypeptides Proteins

• Each of the 20 ? -amino acids found in proteins
  can be distinguished by the R-group substitution
  on the alpha-carbon atom.
• Two broad classes of amino acids based upon
  whether the R-group is hydrophobic or
  hydrophilic.
• The hydrophobic amino acids reside predominantly
  in the interior of proteins. This class of amino
  acids does not ionize nor participate in the
  formation of H-bonds.
• The hydrophilic amino acids tend to interact with
  the aqueous environment, are often involved in
  the formation of H-bonds and are predominantly
  found on the exterior surfaces proteins or in the
  reactive centers of enzymes.

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Amino Acids, Polypeptides Proteins

• Grouping of Amino-Acids
• Aliphatic Chain Amino Acids
• Gly, Ala, Val, Leu, Ile
• Non-Aromatic Amino Acids with -OH
• Ser, Thr
• Amino Acids with Sulfur-Containing R-Groups
• Cys, Met
• Acidic Amino Acids and their Amides
• Asp, Asn, Glu, Gln
• Basic Amino Acids
• Arg, Lys, His
• Amino Acids with Aromatic Rings
• Phe, Trp, Try, Pro

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Amino Acids, Polypeptides Proteins

• Acid-Base Properties of the Amino Acids
• Key Points
• The ?-COOH and ?-NH2 groups in amino acids are
  capable of ionizing (as are the acidic and basic
  R-groups of the amino acids).
• At physiological pH (around 7.4) the carboxyl
  group will be unprotonated and the amino group
  will be protonated.
• An amino acid with no ionizable R-group would be
  electrically neutral at this pH. This species is
  termed a zwitterion.
• As a general rule the amino terminal has a
  pKa9.4 and carboxy-terminal is at pKa2.0

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Amino Acids, Polypeptides Proteins

• The net charge (the algebraic sum of all the
  charged groups present) of any amino acid,
  peptide or protein, will depend upon the pH of
  the surrounding aqueous environment.
• As the pH of a solution of an amino acid or
  protein changes so too does the net charge. This
  phenomenon can be observed during the titration
  of any amino acid or protein.
• When the net charge of an amino acid or protein
  is zero the pH will be equivalent to the
  isoelectric point pI.
• pI(pKa1pKa2)/2

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Amino Acids, Polypeptides Proteins which amino
acid is this?
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Amino Acids, Polypeptides Proteins

• The Peptide Bond
• Amino acids can be joined together to form a
  peptide or polypeptide. They are called peptides
  because when the carboxyl group of one amino acid
  joins to the amino group of another, a peptide
  bond is formed.
• Chemically this is an amide bond but when it
  occurs in proteins it is given the name peptide
  bond.
• The partial double bond nature of the peptide
  bond means that there is not free rotation about
  the C -- N bond. The most stable conformation is
  planar and trans

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Amino Acids, Polypeptides Proteins

• Peptide chain (a.k.a. polypeptide) has direction.
• N-Asparagine-Glutamate-Glycine-C
• What is the pI of this tripeptide? Be sure to
  know the three letter code of all the amino
  acids!
• There is no set length of a polypeptide (how long
  is a piece of string) although most polypeptides
  in nature are between 50 and 2000 residues long.
• So how do we determine the sequence of a
  polypeptide?

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Amino Acids, Polypeptides Proteins

• Prior to sequencing peptides it is necessary to
  eliminate disulfide bonds within peptides and
  between peptides using 2-mercaptoethanol.
• To determine N-terminus
• Sanger Agent 2,4-dinitrofluorobenzene (DNF)
  detected by yellow pigment observed via SDS-PAGE
• Dansyl Chloride Like Sanger however detected via
  Fluorescence.
• Edman Degradation sequential removal of amino
  terminal amino acid using phenylisothiocyanate.
  Now automated.

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Amino Acids, Polypeptides Proteins

• Due to the limitations of the Edman degradation
  technique, peptides longer than around 50
  residues can not be sequenced completely! So we
  have more stuff for you to remember!
• Trypsin cuts carboxyl terminal of LYS, ARG
  except Pro
• Chymotrpsin cuts carboxyl terminal of Aromatic,
  except Pro
• Carboxypeptidase A not specific cuts carboxyl
  terminal of almost aas, except Lys Arg, or if
  Pro is terminal residue
• Carboxypeptidase B not specific cuts carboxyl
  terminal of Lys Arg

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Amino Acids, Polypeptides Proteins

• Other techniques to keep in mind
• Cyanogen bromide (CNBr) This reagent causes
  specific cleavage at the C-terminal side of Met
  residues. The number of peptide fragments that
  result from CNBr cleavage is equivalent to one
  more than the number of Met residues in a
  protein.
• The most reliable chemical technique for
  C-terminal residue identification is
  hydrazinolysis. A peptide is treated with
  hydrazine, NH2-NH2, at high temperature (90oC)
  for an extended length of time (20-100hr). This
  treatment cleaves all of the peptide bonds
  yielding amino-acyl hydrazides of all the amino
  acids excluding the C-terminal residue which can
  be identified chromatographically compared to
  amino acid standards.
• Sample question Answer True/False
• Cynanogen Bromide will cleave this polypeptide
  into a 6-peptide chain and a tripeptide
• Glu-Ser-Thr-Phe-Met-Asn-Trp-Met

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Proteins

• Protein Primary Structure
• The primary structure of peptides and proteins
  refers to the linear number and order of the
  amino acids present. Has all the info to FOLD!
• We can use the methods described earlier to
  determine the sequence. Also see Lecture 4
  PowerPoint for other key factoids and concepts!

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Proteins
Protein Secondary Structure Local
Confirmation 1. Alpha-helix The formation of
the a-helix is spontaneous and is stabilized by
H-bonding between amide nitrogen's and carbonyl
carbons of peptide bonds spaced four residues
apart. Disrupted by Proline 2. Beta sheet
b-sheets are composed of 2 or more different
regions of stretches of at least 5-10 amino
acids. The folding and alignment of stretches of
the polypeptide backbone aside one another to
form b-sheets is stabilized by H-bonding between
amide nitrogens and carbonyl carbons beta-Sheets
are either parallel or antiparallel. In
parallel sheets adjacent peptide chains proceed
in the same direction (i.e. the direction of
N-terminal to C-terminal ends is the same),
whereas, in antiparallel sheets adjacent chains
are aligned in opposite directions. There are
also Super-secondary Structures
(helix-turn-helix, helix-loop-helix and zinc
finger domains of eukaryotic transcription
factors) Do not worry about this much. For the
gunners, Read Voet Voet.
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Proteins

• Tertiary Structure
• See me in 3-D Baby!
• Interaction between amino acid residues
• Hydrophobic amino acids inside
• Hydrophilic amino acids on the surface
• 3-D maintained by hydrophobic, electrostatic
  hydrogen interactions (non-covalent).
• Also present could be disulfide bonds.
• Secondary structures of proteins often
  constitute distinct domains. Therefore, tertiary
  structure also describes the relationship of
  different domains to one another within a
  protein. See the structure of Ig!

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Proteins

• Quaternary Structure
• Many proteins contain 2 or more different
  polypeptide chains that are held in association
  by the same non-covalent forces that stabilize
  the tertiary structures of proteins.
• The intrachain disulfide bond is the one covalent
  bond involved in maintenance of tertiary
  structure.
• The interchain disulfide bond can be used to
  stabilize quaternary structure.

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Carbohydrates

• Carbohydrates are carbon compounds that contain
  large quantities of hydroxyl groups
• carbohydrates that are of physiological
  significance exist in the D-conformation
• Structures you must know! All of them!
• But if nothing else know these
• Monosaccharide
• Disaccharides
• Complex Sugars

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Carbohydrates

• More Structures to Know

L-Fucose is rare L sugar found of the
oligosaccharide chains of N- and O-linked
glycoproteins.
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Carbohydrates
Maltose the major degradation product of starch,
is composed of 2 glucose monomers in an a-(1,4)
glycosidic bond
Lactose is found exclusively in the milk of
mammals and consists of galactose and glucose in
a b-(1,4) glycosidic bond
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Carbohydrates
Sucrose prevalent in sugar cane and sugar beets,
is composed of glucose and fructose through an
a-(1,2)-glycosidic bond
Polysaccharides Carbohydrates found in nature
occur in the form of high molecular weight
polymers called polysaccharides. Ex Glycogen
alpha 1,4 linkage and alpha 1,6 branching of
glucose (animals) Starch amylose only alpha 1,4
linkage of glucose not as compact as
glycogen Amylopectin has branching alpha 1, 6 in
addition to 1,4 linkage. Be aware of the uses of
CHOs in biological systems, Memorize the
glucosaminoglycans pointed out in lecture, know
the structures of O, A, B blood antigens, and how
to distinguish each structure. O is universal
donor, AB universal acceptor, etc.
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Nucleic Acids

• Nucleotides may be considered one of the most
  important metabolites of the cell.
• Nucleotides are found primarily as the monomeric
  units comprising the major nucleic acids of the
  cell, RNA and DNA.
• Used as energy stores ATP, NADH, NADPH, NAD,
  NADP, FAD, FADH2, etc
• cAMP and other second messengers

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Nucleic Acids

• The nucleotides found in cells are derivatives of
  the heterocyclic highly basic, compounds, purine
  and pyrimidine.
• Mnemonic
• CUT pyr (pie) from Pur A G (ag is gold)

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Nucleic Acids
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Nucleic Acids
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Nucleic Acids

• RNA contains A, G, C but has U instead of T

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Nucleic Acids

• Key things to keep in mind for the Exam
• The purine and pyrimidines bases are on the
  inside, while the phosphate and deoxyribose units
  are on the outside of the helix.
• Hydrophobic and van der Waals interactions
  between adjacent base pairs contributes
  significantly to the stability of the helix.
• The two chains are held together by hydrogen
  bonds
• DNA and RNA are read 5?3
• Your answers for questions relating to
  Nucleotides should be in 5?3
• Ex What is the transcript of this DNA sequence?
• a-t-t-g-c-a-g-g-c-c-t-t-a-a-t-g

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Biopolymer Analysis
Techniques Based on Differences in Size
Dialysis Proteins can be separated from small
molecules by dialysis through a semi
permeable membrane Gel Filtration (molecular
sieve chromatography) Used to separate small
molecules from large molecules Small molecules
are slowed, caught in the bead (OPP of Gel
Electrophoresis) Ultracentrifugation This is used
to separate proteins of different sedimentation
coefficients. A high molecular weight molecule
will sediment faster and diffuse slower than a
lower molecular weight molecule of the same
density
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Biopolymer Analysis
Techniques Based on Differences in Size
Dialysis
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Biopolymer Analysis
Techniques Based on Differences in Size
Gel Filtration (Exclusion Chromatography)
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Biopolymer Analysis
Techniques Based on Differences in Size
Gel Electrophoresis This technique is used to
separate proteins, RNA, DNA and Carbohydrates Lar
ge molecules will not move as fast as smaller
molecules (sieving effects of gel) In native gel
electrophoresis where proteins are not denatured
and subunits stay together, the charge of the
protein will come into play in the
separation Sickle cell anemia and sickle cell
trait are diagnosed by a gel electrophoresis
method SDS-PAGE Separate proteins based on MW
alone Proteins denatured and strongly
negative Plot log MW vs. Relative Mobility
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Biopolymer Analysis
Techniques Based on Differences in Size
Gel Electrophoresis
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Biopolymer Analysis
Techniques Based on Differences in Size
Gel Electrophoresis If an antibody-secreting cell
called a plasma cell becomes cancerous, it
grows into a clone secreting a single kind of
antibody molecule. The image shows from left to
right the electrophoretic separation of 1.
normal human serum with its diffuse band of gamma
globulins 2. serum from a patient with multiple
myeloma producing an IgG myeloma protein 3.
serum from a patient with Waldenström's
macroglobulinemia where the cancerous clone 4.
secretes an IgM antibody 5. serum with an IgA
myeloma protein
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Biopolymer Analysis
Techniques Based on Differences in Charge
Ion Exchange Chromatography Negatively charged
molecules will bind to columns of positively
charged beads (DEAE-Sepharose diethylaminoethyl-S
epharose) Positively charged molecules will
bind to columns of negatively charged beads
(CM-Sepharose carboxymethyl- Sepharose) Protein
s can be eluted with increasing salt
concentrations (Na Cl-) because the salt ions
compete with the charged groups for binding to
the charged column
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Biopolymer Analysis
Techniques Based on Differences in Charge
Isoelectric Focusing pI pH at which the
protein has a net ZERO charge Gels containing
polyampholytes of differing pHs are prerun to set
up gradients of pH, then samples are loaded and
electrophoresed until they reach the pH that is
equal to their pI ISOFORMS of proteins can be
identified by this (differ by single amino acids)
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Biopolymer Analysis
Techniques Based on Differences in Affinity
Affinity Chromatography A very specific way to
separate proteins or other molecules that bind to
or are bound by known molecules Create a column
where a specific ligand is conjugated to beads
(like Sepharose or agarose) The sample is run
through the column, unbound material washed away,
and the column is eluted either by competition
(for isolating a receptor on a ligand
column...elute with excess ligand) or by
disrupting binding (for binding that requires
divalentcations, chelate these with EDTA)
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Biopolymer Analysis
Techniques Based on Differences in Affinity
Affinity Chromatography For example, the
antibodies in a serum sample specific for a
particular antigenic determinant can be isolated
by the use of affinity chromatography
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Biopolymer Analysis
Techniques Based on Differences in Affinity
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Biopolymer Analysis
Identification Using Specific Antibodies
ELISA (enzyme linked immunoabsorbant
assay) Allows QUANTIFICATION of a specific
protein in a mixture http//www.biology.arizona.e
du/immunology/activities/elisa/technique.html (ani
mation) Western Immunoblotting Protein mix
separated by SDS page then transferred to a
membrane support which is incubated with a
specific antibody to the protein of interest Then
mixed with a secondary antibody that recognized
the first antibody (this antibody is
radiolabeled) Allows for SPECIFICITY of a
specific protein in a mixture http//www.biology.a
rizona.edu/immunology/activities/western_blot/west
1.html
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Biopolymer Analysis
How would you determine the native molecular
weight of a protein?
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Biopolymer Analysis
How would you determine the native molecular
weight of a protein?
Gel Filtration Ultracentrifugation (using
standards of known MW)
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Biopolymer Analysis
How would you determine the subunit of a
denatured molecular weight of a protein?
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Biopolymer Analysis
How would you determine the subunit of a
denatured molecular weight of a protein?
SDS Page
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Biopolymer Analysis
How would you quantify a protein in a complex
mixture? How would you determine the MW of the
same protein if u had an antibody to it?
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Biopolymer Analysis
How would you quantify a protein in a complex
mixture? How would you determine the MW of the
same protein if u had an antibody to it?
ELISA Western Blot (remember SDS page must be
done first!)
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Enzyme Kinetics
Enzymes are biological catalysts responsible for
supporting almost all of the chemical reactions
that maintain animal homeostasis. Because of
their role in maintaining life processes, the
assay and pharmacological regulation of enzymes
have become key elements in clinical diagnosis
and therapeutics.

• Properties
• Catalytic power
• Specificity for both the reaction catalyzed and
  choice of reactants
• Regulation
• Feedback inhibition
• Regulatory Proteins
• Proteolytic Activation
• Couple Reactions

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Enzyme Kinetics
The favored model of enzyme substrate interaction
is known as the induced fit model. This model
proposes that the initial interaction between
enzyme and substrate is relatively weak, but that
these weak interactions rapidly induce
conformational changes in the enzyme that
strengthen binding and bring catalytic sites
close to substrate bonds to be altered. After
binding takes place, one or more mechanisms of
catalysis generates transition-state complexes
and reaction products
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Enzyme Kinetics
Enzymes increase reaction rates by decreasing the
amount of energy required to form a complex of
reactants that is competent to produce reaction
products. This complex is known as the
transition state complex for the reaction.
Enzymes and other catalysts accelerate
reactions by lowering the energy of the
transition state. The free energy required to
form an activated complex is much lower in the
catalyzed reaction. The amount of energy required
to achieve the transition state is lowered
consequently, at any instant a greater proportion
of the molecules in the population can achieve
the transition state. The result is that the
reaction rate is increased!
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Enzyme Kinetics

• Factors that influence enzyme catalyzed
  reactions
• Temperature
• pH
• Enzyme Concentration
• Substrate Concentration

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Enzyme Kinetics

• At high substrate concentrations the rate
  represented by point C the rate of the reaction
  is almost equal to Vmax

• When the reaction rate becomes independent of
  substrate concentration, or nearly so, the rate
  is said to be zero order.
• Why is point C independent of substrate
  concentration?

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Enzyme Kinetics

• The very small differences in reaction velocity
  at substrate concentrations around point C (near
  Vmax) reflect the fact that at these
  concentrations almost all of the enzyme molecules
  
• are bound to substrate and the rate is virtually
  independent of substrate!
• At lower substrate concentrations, such as at
  points A and B, the lower reaction velocities
  indicate that at any moment only a portion of the
  enzyme molecules are bound to the substrate

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Enzyme Kinetics

• In fact, at the substrate concentration denoted
  by point B, exactly half the enzyme molecules are
  in an ES complex at any instant and the rate is
  exactly 1/2 Vmax. At substrate concentrations
  near point A the rate appears to be directly
  proportional to substrate concentration, and the
  reaction rate is said to be first order.

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• Km
• The substrate concentration at which the
  velocity of the reaction
• is half the maximum velocity.
• A substrate with the lowest Km value for an
  enzyme
• has the highest apparent affinity for that enzyme

Turnover number Vmax/ET k2 Moles of substrate
transformed per second per mole of active site
The best substrate for an enzyme is that which
has the highest Vmax/Km
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Enzyme Kinetics
To avoid dealing with curvilinear plots of enzyme
catalyzed reactions, biochemists Lineweaver and
Burk introduced an analysis of enzyme kinetics
based on the following rearrangement of the
Michaelis-Menten equation Plots of 1/v
versus 1/S yield straight lines having a slope
of Km/Vmax and an intercept on the ordinate at
1/Vmax
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Enzyme Kinetics
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Enzyme Kinetics
Noncompetitive Inhibitor Km unchanged Vmax
decreased (appear as if less enzyme)
Competitive Inhibitor Km increased Vmax
unchanged
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Enzyme Kinetics
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Enzyme Kinetics

• In Enzyme Kinetics III there are some major
  points to understand
• Cooperativity
• Sigmoidal vs. Hyperbolic Curves
• Allosteric Enzymes and their binding proteins
  (inhibitors vs. activators)
• Homotropic and Heterotropic interactions

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Enzyme Kinetics
Allosteric effectors bring about catalytic
modification by binding to the enzyme at distinct
allosteric sites, well removed from the catalytic
site, and causing conformational changes that are
transmitted through the bulk of the protein to
the catalytically active site(s).
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Bioenergetics
Why is bioenergetics important to us?
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Bioenergetics
Thermodynamics
Energy changes in a system are governed by laws
of thermodynamics Thermodynamics is the study of
the patterns of energy change The "thermo"
refers to energy, and "dynamics" means patterns
of change More specifically, thermodynamics
deals with (a) energy conversion and (b) the
stability of molecules and direction of change
First law Energy can neither be created nor
destroyed, only converted into different
forms Second law All processes progress toward
a state of maximum randomness or disorder
(entropy)
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Bioenergetics
Entropy (S) is the energy in a system that is
unavailable to do useful work Free energy (G) is
the energy in a system available to do useful
work dG dH - TdS (T absolute temperature in
Kelvin) We use dG to express differences in the
free energy of a reaction at ANY concentration
not just equimolar or 1M
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Bioenergetics
dG dG0 RT ln (products/reactants) At
equilibrium dG 0, therefore dG0 -RT ln Keq
or -2.3 RT log Keq
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Bioenergetics
The reaction fructose 6-P ? fructose
1,6-bisphosphate H20 has a Keq of 0.001 _at_ a
pH 7. What is the dGo for this reaction?
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Bioenergetics
The reaction fructose 6-P ? fructose
1,6-bisphosphate H20 has a Keq of 0.001 _at_ a pH
7. What is the dGo for this reaction?
dGo - RT ln Keq - (298)(1.987) ln
(0.001) 4090.26 cal/mol
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Bioenergetics
The reaction fructose 6-P ? fructose
1,6-bisphosphate H20 has a Keq of 0.001 _at_ a pH
7. What is the dGo for this reaction?
dGo - RT ln Keq - (298)(1.987) ln
(0.001) 4090.26 cal/mol
Does this reaction proceed forward?
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Bioenergetics
Does this reaction proceed forward?
Exergonic reaction Any process that exhibits a
negative free energy change proceeds to
equilibrium and energy is given off Endergonic
reaction A process that exhibits a positive free
energy change cannot proceed independently and
energy from another source must be provided
When Keq lt 1, the reaction is endergonic and dG
is positive When Keq gt1, the reaction is
exergonic and dG is negative
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Bioenergetics

• Different tissues can have different values of dG
  for the hydrolysis of ATP.
• In muscle cells, ATP 8.1 mM, ADP 0.93 mM
  and Pi 8.1 mM.
• In RBCs , ATP 2.3 mM, ADP 0.3 mM and Pi
  1.7 mM. Which tissue requires the most energy
  to synthesize ATP and derives the most energy
  from the hydrolysis of ATP?

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Bioenergetics
Which tissue requires the most energy to
synthesize ATP and derives the most energy from
the hydrolysis of ATP?
?G ?Go - 2.3 RT log Pi ADP / ATP
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Bioenergetics

• Other important concepts
• dG of high energy compounds
• ATP -7.3
• Creatine phosphate -10.3
• Acetyl CoA -7.7
• Hesss Law
• As long as the sum of all the reactions in the
  pathway give a negative value, the reaction is
  thermodynamically favorable
• Free energy change and redox
• dG -nFE
• Most negative E is best reductant, most willing
  to give up an electron (ie NADH)
• Most positive E is best oxidant, most willing to
  accept an electron
• (ie 02)

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Study Questions

• You have found that there are elevated levels of
  a specific enzyme in the serum of patients in the
  early stages of emphysema. It happens that this
  enzyme exists in two isoforms, A and B, that have
  the same molecular weights but differ slightly in
  their amino acid sequences. It seems that the B
  isoform predominates in the lung whereas the A
  isoform is found in most other tissues. You need
  to find a way to distinguish these two isoforms
  so you can distinguish lung tissue damage from
  other tissue damage. Which protein separation
  technique would you try first?
• a) Ultracentrifugation
• b) Gel filtration
• c) Nondenaturing or native gel electrophoresis.
• d) SDS-polyacrylamide gel electrophoresis
  (SDS-PAGE)
• e) Western immunoblot analysis
• NOTE What other method could you use?

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Study Questions

• Immunological methods (those using specific
  antibodies) are very important diagnostic tools
  for the detection of specific proteins and
  carbohydrates (antigens) in the serum of patients
  with a variety of diseases. Which immunological
  method would be the fastest way to determine
  whether a particular antigen is present in a
  patient's serum?
• a) Isoelectric focusing
• b) Immunofluorescence microscopy
• c) ELISA (Enzyme linked immunoabsorbant assay).
• d) Immunoprecipitation of the radiolabeled
  protein and analysis by SDS polyacrylamide gel
  electrophoresis

84
Study Questions

• The Km of an enzyme reaction can tell us several
  things about the reaction including
• a) The affinity of the enzyme for a particular
  substrate. The higher the Km, the lower the
  enzyme's affinity for that substrate.
• b) The effective concentration of the enzyme in
  vivo
• c) The turnover number or efficiency, of the
  enzyme catalyzed reaction
• d) The enzyme concentration at one half the
  maximum velocity of the reaction
• e) The energy of activation of the enzyme
  catalyzed reaction

85
Study Questions

• You are comparing the properties (Vmax and Km) of
  two substrates for one enzyme. Your experiments
  show that the enzyme has a Vmax of 1000 and a Km
  of 10 for Substrate A and a Vmax of 25000 and a
  Km of 1000 for Substrate B. With which substrate
  does the enzyme work more efficiently and why?
• a) Substrate B because the Vmax is higher with
  that substrate
• b) Substrate A because the Km is lower for that
  substrate
• c) Substrate B because the Km is higher for that
  substrate
• d) Substrate A because the VmaxKm ratio is
  higher.
• e) Substrate B because the VmaxKm ratio is lower

86
Study Questions

• Choose the INCORRECT statement concerning
  allosteric enzymes and proteins
• a) Allosteric enzymes are those which are
  regulated by effectors that bind at sites other
  than the enzymes active site
• b) Allosteric enzymes do not follow Michaelis
  Menten kinetics and have sigmoidal velocity
  versus substrate curves
• c) Allosteric enzymes are generally multi-subunit
  proteins that also exhibit cooperativity
• d) An allosteric activator shifts the velocity
  versus substrate curve to the right indicating
  the enzyme now has a higher Km.

87
Study Questions

• Deficiency of muscle phosphorylase (McArdles
  disease) results in an inability to perform
  exercise. The standard free energy ?G0 for
  phosphorylase shown below, is 3.3 kcal/mole
• (glucose)n H3PO4 lt-gt (glucose)n-1
  glucose-1-phosphate
• Which of the following statements can be deduced
  about the above reaction, from the data given?
• a) The equilibrium constant, Keq is greater than
  1
• b) The reaction is endergonic under standard
  conditions.
• c) In the cell the reaction proceeds from left to
  right only in the presence of ATP
• d) The velocity of the reaction is independent of
  the presence of the enzyme, phosphorylase
• e) The reaction is responsible for glycogen
  synthesis in vivo, because it can only go from
  right to left

88
Study Questions

• Molecular oxygen
• a) is a good electron acceptor.
• b) donates electrons to many acceptors
• c) is an essential component of all oxidations
• d) has less entropy, than water
• e) is reduced to water only at the expense of 3
  ATPs

89
Study Questions

• Identify the oxidant and reductant in this
  reaction catalyzed by succinate dehydrogenase
• Malate NAD ? Oxaloacetate NADH H
• a) NAD is the oxidant and Malate is the
  reductant.
• b) NAD is the reductant and Malate is the
  oxidant
• c) Oxaloacetate is the reductant and NADH is the
  oxidant
• d) It is impossible to determine using just this
  equation
• e) NAD is the reductant and NADH is the oxidant

90
GOOD LUCK!

• Histo review next Wednesday, Room 423
• 4-6pm.
• MIKE BROMAN (MD/PhD candidate).
• HIGHLY RECOMMEND ATTENDING!