skeleton notes

Introduction to biological chemistry, Redinbo, M.R.

Chapter 1: Life

I. Introduction to Biological Chemistry

A. Biochemistry literally means the study of the chemistry of life

B. Life expresses itself through biomolecules

II. Types of biomolecules—many are polymers of repeating subunits

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III. Questions to ask yourself as a biochemist:

A. What are the chemical and 3-dimensional structures of biomoleculesB. How do biological macromolecules interact with eachotherC. How are biomolecules synthesized and degradedD. How does the organization of biomolecules influence their activity

VI. Biology obeys the laws of thermodynamics

A. Physical forces influence the behavior of biological systems

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Chapter 2: Water

Read prior to lecture

2.1 Weak Interactions in Aqueous Systems

Hydrogen bonding gives water its unusual properties Water forms Hydrogen bonds with polar solutes Water interacts electrostatically with charged solutes Entropy Increases as crystalline substances dissolve Nonpolar gases are poorly soluble in water Nonpolar compounds force energetically unfavorable changes in

the structure of water Van der Waals interactions are weak interatomic attractions

Guided reading questions:1. what are the structural and physical properties that make water

ideal as a solvent for life? List a few characteristics.2. How strong are Hydrogen bonds and how do they contribute to

water’s physical properties3. What is Entropy and why, in thermodynamic terms, does it

dictate the solubility/insolubility of molecules and gases? How does this relate to the structure of water.

4. Why are Van der Waals forces critical for micelle formation and what is the role of entropy in the process

5. Define in your own words Ka, pKa, and pI

I. Physical properties of water

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II. Structure of water

A. Water has Hydrogen bonding (H-bond.) potential

1. H-bond: Noncovalent interaction in which a H is partially shared between 2 electronegative atoms

B. Water is a rapidly moving collection of H-bonds

C. H-bonding plays an important role in the interaction of biomolecules

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III. Role of water as a solvent

A. Interaction with polar solutes: Does overall entropy for this process increase or decrease? Is S positive or negative?

B. Interaction with nonpolar solutes: Does overall entropy for this process increase or decrease? Is S positive or negative?

C. Interaction with amphiphillic solutes: Where is entropy increasing and where is there a decrease in entropy? Why do these spontaneously form in vivo?

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In class review of titrations

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CHAPTER 3: AMINO ACIDSRead Prior to class:3.1 Amino Acids

Amino acids share common structural features The amino acid residues in proteins are L stereoisomers Amino Acids can be classified by R group Amino acids have characteristic titration curves Titration curves predict the electric harge of amino acids Amino acids differ in their acid-base properties

Omit uncommon amino acids

Guided reading questions Chapter 3 and 41. What are the acid/base characteristics of the 20 amino

acids used to determine charge at a given pH2. How are the amino acids classified? Using table 3-1 in

Lehninger, what do each of the pK values tell you about the acid/base characteristics of an amino acid? Why do some have 2 and others 3

3. Can one amino acid belong to more than one subcategory? Cite an example (i.e. both acid and aromatic?)

4. Redefine pKa in light of the terms pK1, pK2 and pKr. How do you find the pI of an amino acid with a nonpolar R-group versus one with an acidic R-group?

5. Why is the pK1 value of glycine different from that of acetic acid (both contain a single –COO- function attached to a single carbon)

6. What kind of bond is the peptide bond? What is a condensation reaction?

7. What role does resonance play as it relates to the peptide bond in polypeptides?

I. Introduction and Background

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A. Biochemists have been studying proteins since the 1800’s

B. Abundance in cell

WaterProteins

Nucleic AcidsCarbohydrates

LipidsOther

C. Proteins play a role in just about every biological process

1.

2.

3.

4.

II. Proteins are built from Amino Acids

A. General structure of Amino Acids and factors influencing charge

Can you identify and mathematically express each of the following important points in the titration curve:

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pK1:

pK2:

pI:

B. The titration curve of Lysine

Draw the species that predominates below pK1

Draw the species that predominates above pK1

Draw the species that predominates below pKR

Draw the species that predominates above pKR

What species would predominate at pH 9.5

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III. Amino Acids, the Peptide Bond, and Peptides

Draw the condensation reaction resulting in a dipeptide (use R1 and R2 as the side chain R groups) and the resonance structures of the peptide bond.

Recall the geometry about atoms involved in amide linakages. Place a box around the atoms that form a rigid plane and are coplanar on your diagram above

A. Peptides and proteins have directionality

NH3+ --------------------------------------------------------------COO-

1.

2.

Calculating the pI of a polypeptide—In class group assignment

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(a)(b) Using the stylized format on the right and table 3-1, draw which species of the

peptide exists at pH 1, pH 3.5, pH 4.2, pH 8.5, pH 10, and pH 12.(c) Would Lys-Glu-Ser have the sam pI as Ser-Glu-Lys?(d) What is the pI of Lys-Glu-Ser?

IV. Characteristics of the peptide bond

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A. How can an amide plane rotate? Is it possible?

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B. Certain combinations of and are forbidden

1. Why? Each and appear to have 360o of free rotation.

C. What are the allowed combinations of and ?

CHAPTER 4: AMINO ACIDS, POLYPEPTIDES, AND HIGHER LEVELS OF PROTEIN ARCHITECTURE.

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Read prior to class:4.1 Overview of Protein Structure

A protein’s conformation is stabilized largely by weak interactions The peptide bond is rigid and planar

4.2 Protein Secondary Structure

The alpha Helix is a common Protein Secondary Structure Amino Acid Sequence Affects stability of the alpha Helix The Beta Conformation organizes polypeptide chains into sheets Skip Beta turns Common secondary structures have characteristic dihedral angles

4.3 Protein Tertiary and Quaternary Structures

Fibrous proteins are adapted for a structureal function (focus on alpha keratin) Skip ahead to: globular proteins have a variety of tertiary structures (stop after

reading about motifs, folds, supersecondary structures, and domains)

4.4 Protein Denaturation and Folding

Amino acid sequence determines tertiary structure Polypeptides fold rapidly by a stepwise process. Some proteins undergo

assisted folding (only 1st two paragraphs and figure 4-40, Chaperones in protein folding)

Guided reading1. List and describe 5 forces that stabilize protein 3-D structure2. How do amide planes and primary sequence influence the possible

conformations of a fully folded protein?3. What are the common stabilizing forces of the two main types of secondary

structures? 4. How is the H-bonding pattern in the alpha helix similar to the H-bonding

pattern in the beta-sheet?5. What is the H-bonding pattern in the alpha helix? How many residues/turn

in an alpha helix?6. What are the differences and similarities between parallel and antiparallel

beta sheets?7. Define in your own words “tertiary and quaternary structure”8. What are domains and how are they different from motifs?9. In figure 4-26, why is the thermal denaturation curve of Ribonuclease A left-

shifted compared to Apomyoglobin? Why is the curve sigmoidal?10. How did Anfinsen’s experiment “solve” Levinthal’s paradox?

I. Levels of structural organization

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More details about:

a.) Primary structure:

example: 60 residue protein and we have 20 naturally occurring amino acids. What is the total possible number of combinations of primary sequence for this protein?

b.) Secondary structure:

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1.) Alpha helix: repeating pattern of weak forces = stability

In Class Group Problem:

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2.) Beta Sheet

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c.) Tertiary structure:

Tertiary structure determined by:

1.

2.

3.

4.

5.

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General characteristics of a fully folded protein:

d.) Quaternary structure:

1.

Example: We will examine hemoglobin in much more detail later in this class

II. Protein Folding

example: Cyrus Levinthal 100 residue protein.

2. Proposed sequence of events in protein folding

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Compare the four folding funnels above. What information can we glean about the folding process in terms of stability and intermediates as they all head to the N state? If proteins fold spontaneously, what does that mean with respect to S and G?

D. In vivo—how do proteins fold properly?

In class group problem:

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Draw a reaction coordinate diagram for a protein starting in the unfolded state and ending in the folded state. Your X-axis should be reaction progress and your Y-axis should be energy. (Think back to general/organic chemistry and transition state theory)

III. Denatured (unfolded) proteins can be refolded

A. Ways to unfold and/ or precipitate proteins

In class example:

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%proteins folded in native state

[Urea]

%proteins in solution

[Ammonium Sulfate]


Examine the two graphs below. Both Urea and Ammonium Sulfate are in the family of “Chaeotropic Salts”. (a) Explain why the nature of the Urea vs. Native protein plot in molecular terms, i.e. how is urea affecting the protein to give us these data. (b) Examine the % proteins in solution vs. Ammonium Sulfate plot. What is the chemical nature (use words like: pI, amino acid composition, hydrophilic, hydrophobic, polar, etc.) of the proteins that begin to precipitate early (see arrow #1)? What is it about the proteins at arrow #2 that prevent them from every coming out of solution?

a.

b.

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Famous experiment by Christian Anfinsen – RNase A

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Protein purificationRead Prior to class:3.3 Working with proteins

Proteins can be separated and purified Proteins can be separated and characterized by electrophoresis

(omit purification tables, isoelectric focusing, and 2-D electrophoresis)

Guided reading questions:1. How does a high vs. low [salt] affect protein solubility? Will all proteins have

the same solubility properties at a given [salt]?2. How do you know if a protein is positively or negatively charged just by

knowing its pI value?3. Have a functional and working understanding of the differences between:

Ion-exchange chromatography, size exclusion chromatography, and affinity chromatography

4. What is the chemical importance of SDS in visualizing proteins by SDS-PAGE?

5. How do proteins migrate (separate) in an electrophoretic field via SDS-PAGE

I. Characteristics of Proteins

A. Every Protein has a unique primary sequence. Hence, every protein has several unique characteristics

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III. Methods of cell disruption

A. There are a number of ways to prepare a protein extract from prokaryotic and eukaryotic cells.

II. Protein Purification Methods Review

A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties:_____________________pI Size M r Bind to DNA?

protein A 7.4 82,000 yesprotein B 3.8 21,500 yesprotein C 7.9 23,000 noprotein X 7.8 22,000 yes_________________________________________________________

Briefly explain (1 or 2 non-run-on sentences) what type of protein separation techniques might she use to separate

(a) protein X from protein A?

(b) protein X from protein B?

(c) protein X from protein C?

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III. Visualizing Proteins: Proteins are charged; therefore, they will migrate in an electric field.

Problem: Will acidic and basic proteins migrate in the same manner?

A. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

Structure of polyacrylamide at a molecular level: http://www.ucl.ac.uk/~ucbcdab/enzpur/SDS-PAGE.htm

A protein has been shown to have a molecular weight = 140,000g/mol. You observe the following when your run your SDS-PAGE both in the absence of BME (lane A) and the presence of BME (lane B). Lane C is a molecular weight marker. Using simple geometric symbols, draw the native protein in terms of number of subunits, stoichiometry of subunits, and the kinds of bonding (covalent: use –s-s-, noncovalent: use ≈ ) that exist.

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PROTEIN FUNCTION

Read prior to class:5.1 Reversible Binding of a Protien to a Ligand: Oxygen-Binding Proteins

Oxygan can bind to a Heme prosthetic group globins are a family of oxygen-binding proteins myglobin has a single binding site for oxygen protein ligand interactions can be described quantitatively (know

what theta is, but you don't need to derive eqtn 5-4 and 5-5) proteins structure affects how ligands bind Hb transports O2 in blood Hb and Mb are structurally similar Hb undergoes a structural change on binding O2

Hb binds O2 cooperatively---stop once you read the definition of allosteric protein (no homotropic/heterotropic control, hill plots, etc.

Hb also transports H+ and CO2 (Bohr effect in here, we will not cover CO2 binding)

O2 binding to Hb is regulated by BPG Sickle-cell anemia is a molecular disease of Hb

Guided reading questions:1. Compare and contrast the structural and functional similarities between

Myoglobin (Mb) and Hemoglobin (Hb). Make a list.2. How does the Mb O2 binding curve differ from that of Hb’s O2 binding

curve? What does the shape of each curve tell us about ligand binding?

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What does low affinity versus high affinity mean in terms of p50 values on a theta plot (theta on Y and pO2 on X-axis)

3. Define for yourself “allosterism”, “cooperativity”, “dynamic equilibrium”, and “conformational state” as they relate to Hb.

4. How do the T and R states of Hb differ both in overall structure and O2 affinity?

5. What is the role of pO2 in oxygen transport from lung to tissue?6. What is the Bohr effect? What are the Bohr protons and how do they

stabilize modulate T and R state interconversion? What role does pH play in oxygen transport from lung to tissue?

7. What is BPG? Where does it bind to Hb? What forces stabilize its binding? What role does BPG play in oxygen transport from lung to tissue?

8. Why does a single mutation in Hb lead to such a dramatic pathology? Are there other Hb mutations that have little to no disease associated?

We will use the examples of Myoglobin and Hemoglobin to discuss how protein tertiary and quaternary structure influence function.

A. Myoglobin:

B. Hemoglobin:

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C. Cooperativity, Conformational changes, and oxygen binding in Hemoglobin

1. Hemoglobin has two conformational states:

T-state

R-state

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Hb has two conformational states – The Bohr effect

A. Binding of O2 to Hb stimulates the release of H+ from Hb (the Bohr effect)

Graph time: O2 binding by Hb at different pH values in vitro

Group in class problem:When you are working late in lab one night, you discover: in oxygenated Hb, the pKr for the His residues at position 146 of the Beta chain is 6.6. In deoxygenated Hb, the pKr = 8.2 for the same His residue (His 146 of the Beta chain). Describe these findings focusing on the Bohr effect and the conformational states of Hb.

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2,3-bisphosphoglycerate (BPG) modulates the conformational changes within Hb

A. Highly purified (“stripped”) Hb has a higher affinity for O2 than Hb in whole blood (similar to Mb !!!)

B. Blood must contain some other compound that affects O2 binding to Hb

C. The metabolite BPG is that compound.

Graph time: make a theta plot of stripped Hb and blood Hb. What purpose does BPG serve?

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A new O2 transport protein has been discovered. The deoxy protein structure reveals a dimeric (homodimer) structure shown below in figure a. The individual monmeric

subunits interact via salt bridges between the N- and C-termini. Helices A and C are stabilized by a salt bridge between Histidine 13 and Aspartic acid 85. The O2 site is between the rigid linked helices A and C (see figure b). In the deoxy state, the space between the Fe2+ atoms is too small to bind O2, thus the atoms must be forced apart when O2 is bound

Answer the following questions, briefly explaining your answer in terms of the structure

(a) Will this molecule show cooperative oxygen binding?

(b) Will this molecule exhibit a Bohr effect

(c) Would the addition of BPG have an effect on O2 binding

(d) What would be the effect of a mutation which replaced aspartic acid 85 with a Lysine residue (pKR = 10.53)

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2. A single mutation in hemoglobin can lead to altered structure and function (Sickle Cell Anemia).

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Chapter 6: ENZYME CATALYSIS

Read Prior to class:6.1 Intro to enzymes

most enzymes are proteins enzymes are classified by the reactions they catalyze

6.2 how enzymes work,

enzyme affect reaction rates, not equilibria reaction rates and equilibria have precise thermodynamic

definitions a few principles explain the catalytic power and specificity of

enzymes weak interactions between enzyme and substrate are optimized

in the transition state binding energy contributes to reaction specificity and catalysis

6.3 enzyme kinetics as an approach to understating mechanism

Substrate concentration affects the rate of enzyme catalyzed reactions

The relationship between substrate concentration and reaction rate can be expressed quantitatively,

Kinetic parameters are used to compare enzyme activities (1st paragraphs and turnover number and M-M equation)

NO: catalytic efficiency or specificity constant Skip ahead to: enzymes are subject to reversible or irreversible

inhibition. Focus on: Competitive inhibition AND under ‘mixed inhibitor’ you

will find noncompetitive inhibition. Competitive and noncompetitive are the only two we will cover (no noncompetitive or all mixed)

6.4 examples of enzymatic reactions

The chymotrypsin mechanism involves acylation and deacylation of a Ser residue

NOTE: don’t memorize the reaction on p. 217-ish, be able to follow where you see the different types of catalytic mechanisms…covalent catalysis, acid base, etc…

ONLY DO THE END OF CHAPTER QUESTIONS THAT ARE ASSOCIATED WITH THESE TOPICS.

Guided reading questions:

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1. What kinds of molecules are enzymes and why are they necessary for biochemical reactions in the cell?

2. Thermodynamically, how do enzymes catalyze reactions? What do they change and what do they NOT change (fig. 6-2)

3. What is the difference between a binding pocket and an active site?4. Define in your own words: “binding energy” and “induced fit”5. What are the specific types of catalysis that enzymes utilize? List and define

three from your book.6. (For next lecture, “kinetics” What were the assumptions Miichaelis and

Menten made when deriving their equation? What is Km mathematically and conceptually?

I. General properties of enzymes

Enzymes: catalysts in biological systems that increase the rate of reactions, but are not changed at the end of the process

Enzymes allow for:

1.

2.

3.

4.

II. The six general classes of enzymes

A.ENZYME CLASS TYPE OF REACTION1. Oxidoreductases Redox (e- transfer reactions)2. Transferases Transfer functional group3. Hydrolases Hydrolysis (break bond with H2O)4. Lyases Add or remove groups to make or break

C=C bonds5. Isomerasses Rearrange C-skeleton6. Ligases Form: C-C; C-N; C-O; C-S bonds at the

expense of ATP hydrolysis to ADP

B. Enzymes as catalysts:

1. Enzymes do not change the ______ for a reaction

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2. Enzymes alter the _______ of a reaction

Example: Reaction coordinate diagram

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III. Catalytic Mechanisms: factors contributing to enzymes’ catalytic activity. Note: enzymes may use one or more of these general strategies.

a. Proximity and orientation

b. Preferential transition state binding

c. General Acid-Base chemistry

d. Covalent catalysis

e. Electrostatic catalysis

f. Metal ion catalysis

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IV. Catalytic mechanism--chymotrypsin

A. The catalytic mechanism of chymotrypsin. A long winded guide to understanding catalytic mechanisms. Enjoy the show.

Group exercise:The figure below shows one of the possible reaction mechanisms for the enzyme, Lysozyme. Looking at the panels from left to right, List at least 4 types of enzymatic mechanisms/strategies demonstrated in this figure? Please state your answer in the form, “Glu35 is demonstrating ________ catalysis, and Asp52 is demonstrating…etc”. You can also refer to the entire enzyme if it is demonstrating a catalytic strategy as a whole.

Solving enzyme structure function problems:The structure of the enzyme Tryptophan Synthetase has been studied extensively by a variety of methods. In a series of studies, Yanofsky and co-workers examined the effect on enzyme activity of various amino acid changes in the protein sequence (Federation Proceedings, 22:75 (1963) and Science 146:1593 (1964)). Altered amino acids are shown in bold. "Wild-type" is the normal strain isolated from the wild.

Postulate TWO possible explanations for these data1.2.

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Yanofsky & co. collected more mutants and examined their proteins to determine which of the above explanations was more likely to be correct:

part 2. Which of your two models is supported by these data? explain? Alterations of amino acids at another location in the protein were found to interact with alterations at position A.

part 3. Explain the behavior of mutant 6 in terms of your model of part b.

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ENZYME KINETICS, INHIBITION, AND REGULATION

IV. II. Enzyme catalyzed reactions in biological systems

A. For a simple enzyme catalyzed reaction:

S = substrate and P = products

B. Biological reactions are generally more complex

1. S1 + S2 P2. S P1 + P2

3. S1 + S2 P1 + P2

In class group excerciseFor reaction 1: What is the equilibrium constant for this reaction (not the number but Keq = ?)Where do the rate constant(s) for this reaction go?Can you write a rate equation for this reaction

C. In the simple case of S being converted to P in an enzyme catalyzed reaction, the enzyme [E] must form a complex with substrate [S] to yield an enzyme-substrate complex [ES] in order to form product [P].

I. Michaelis and Menten (M & M): Their pioneering work on enzyme kinetics began around 1913

A. M & M began investigating the effects of [S] on the formation of the ES complex. They examined the effects by measuring the initial reaction velocity (o).

Question: What if we keep the [E] constant and vary the [S]?

Simple Concept Example:

E + S ES P

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II. Derivation of the M & M equation:

Assumption 1 (assumption of equilibrium): Early in the reaction, little P has accumulated so k-2 can be ignored.

P/time = (o) = k2 [ES]

Eqtn. 1 Formation of ES: rate = k1 [E] [S]

Eqtn. 2 Breakdown of ES: rate = k-1 [ES]

rate = k2 [ES]

Steady State Assumption: Once reaction gets started, the [ES] remains constant. As a result, the formation of ES must equal the Breakdown of ES:

Eqtn. 3

Eqtn. 4

Question: Knowing this, write a mathematical expression showing the Steady State Assumption and then show it graphically. X-axis is reaction progress, Y-axis is amount or concentration. Show E, S, and ES

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Now rewrite this expression in terms of ES:

Eqtn. 5 Define Km

…lots of algebra (see your text or online resources) for eqtns. 6-9

“Ta Da!”, the Michaelis-Menten equation is:

Eqtn. 10

SO WHO CARES?

Consider the special case when o = ½ Vmax

Eqtn. 11

Use this space to get from eqtn. 11 to 15 (hint: get rid of Vmax first)

Eqtn. 15

When o = ½ Vmax, Km = [S] that gives ½ Vmax

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III. Importance of K m and Vmax

A. Km is a measure of an enzymes “affinity” for its substrate.

B. Km is unique for each enzyme-substrate pair

1. Enzyme with high Km

2. Enzyme with low Km

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KMVmax

1[S] + 1

Vmax

= 1vi


C. Determining Km and Vmax experimentally

1. Difficult to determine Km and Vmax from:

2. Lineweaver-Burk or Double Reciprocal Plot. (think mx+b = y)

Y-intercept =

X-intercept =

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Using Km, Vmax, and kcat in practice

a. k cat = # of rxns an enzyme can carry out; most commonly the rate determining step in an enzyme catalyzed reaction. This is also referred to as Turnover number = Vmax/[E total] (units = mins-1 or s-1)

The enzyme dihydrolipoyl transferase is absolutely required for aerobic respiration. It is a large enzyme with a M.W. = 120,000g/mol. It enzymatically transfers an acetyl group from pyruvate to Coenzyme A in the eukaryotic mitochondrion. If 40 μg of pure dihydrolipoyl transferase catalyzes the transfer of 1.2g of substrate to product (M.W. = 87.0 g/mol) in 4 minutes while functioning at Vmax, what is the turnover number (kcat) of this enzyme in units of min-1?

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IV. Enzyme Inhibition

A. Competitive Inhibition

Effects of a competitive inhibitor on Km and Vmax:

Effects of a noncompetitive inhibitor on Km and Vmax:

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skeleton notes

Documents

role of water

stereoisomers amino

water ideal

polar solutes water

physical properties

amino acidsread

r group amino acids

structure of water van