BioChem Handouts Part 1 (EAmor)

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Introduction
On Life and Chemistry..

Living things are composed of lifeless molecules (Albert Lehninger) Chemistry is the logic of biological phenomena (Garrett and Grisham) What on earth is not biochemistry? (Anonymous)
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Scope of this Review

Proteins Carbohydrates Lipids Nucleic Acids Metabolism
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Polymer of L-amino acids Have varied roles most important of which is catalysis and regulation
Polymer of monosaccharides
Main source of energy in most organisms
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nformational i
molecules of all living organisms tructural and functional parts s of units such as the ribosome atalytic function (ribozymes) c ransport and provide t chemical energy in the form of phosphate groups mportant in cell regulation i and signal transduction
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The language of nucleic acids
What makes us human

There are 46
chromosomes, arranged in 23 pairs, in each cell in the body
One chromosome of
each pair is contributed by the father, and the other by the mother
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Biochemistry for the MED Boards
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Distinctive Properties of Living Systems

Organisms are complicated and highly organized Biological structures serve functional purposes Living systems are actively engaged in energy transformations Living systems have a remarkable capacity for self-replication
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The sequence of monomeric units in a biological polymer has the potential to contain information if the diversity and order of the units are not overly simple or repetitive. Nucleic acids and proteins are information-rich molecules; polysaccharides are not.
IN008
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Methods for Separating and Purifying Biomolecules

Salt fractionation (precipitation of proteins with ammonium sulfate) Chromatography paper, ion-exchange, affinity, thin-layer, gas-liquid, high pressure liquid, gel filtration Electrophoresis paper, high voltage, agarose, cellulose acetate, starch gel, polyacrylamide gel, SDS-PAGE Ultracentrifugation
Methods for Determining Biomolecular Structures

Elemental analysis UV-VIS, IR, NMR spectroscopy Acid/base hydrolysis Enzymatic degradation MS Specific sequencing methods X-ray crystallography
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Preparations for Studying Biochemical Processes

Whole animal (transgenic and with gene knockout) Isolated perfused organ Tissue slice Whole cells Homogenate Isolated cell organelles Subfractionation of organelles Purified metabolites and enzymes Isolated genes (PCR and site-directed mutagenesis)
Major Causes of Diseases

All of the causes listed act by influencing the various biochemical mechanisms in the cell or in the body.
Physical agents mechanical trauma, extremes of T, sudden changes in atmospheric P, radiation, electric shock
Chemical agents, including drugs: certain toxic compounds, therapeutic drugs, etc. Biologic agents: viruses, bacteria, fungi, higher forms of parasites
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Major Causes of Diseases

Oxygen lack: loss of blood supply, depletion of the oxygen carrying capacity of the blood, poisoning of the oxidative enzymes Genetic disorders congenital, molecular Immunologic reactions anaphylaxis, autoimmune disease Nutritional imbalances deficiencies, excesses Endocrine imbalances hormonal deficiencies, excesses
Amino Acids
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What Are the Structures and Properties of Amino Acids, the Building Blocks of Proteins?
Amino acids contain a central tetrahedral carbon atom There are 20 common amino acids Amino acids can join via peptide bonds Several amino acids occur only rarely in proteins Some amino acids are not found in proteins
Amino Acids Building Blocks of Proteins
Anatomy of an amino acid. Except for proline and its derivatives, all of the amino acids commonly found in proteins possess this type of structure.
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20 Common Amino Acids

You should know names, structures, pKa values, 3-letter and 1-letter codes Non-polar amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids
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The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain. The cationic form is the low pH form, and the titration of the cationic Biochemistry for the MED Boards species with base yields the zwitterion and finally the anionic form.
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pKa Values of the Amino Acids

Alpha carboxyl group - pKa = 2 Alpha amino group - pKa = 9
Arginine, Arg, R: pKa(guanidino group) = 12.5 Aspartic Acid, Asp, D: pKa = 3.9 Cysteine, Cys, C: pKa = 8.3 Glutamic Acid, Glu, E: pKa = 4.3 Histidine, His, H: pKa = 6.0
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Lysine, Lys, K: pKa = 10.5 Serine, Ser, S: pKa = 13 Threonine, Thr, T: pKa = 13 Tyrosine, Tyr, Y: pKa = 10.1
Titration of glycine, a simple amino acid. The isoelectric point, pI, the pH where the molecule has a net charge of 0, is defined as (pK1+ pK2)/2.
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Titration of glutamic acid.
Titration of lysine.
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A Sample Calculation
What is the pH of a glutamic acid solution if the alpha carboxyl is 1/4 dissociated? H = 2 + log10 [1] p [3] H = 2 + (-0.477) p H = 1.523 p
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Reactions of Amino Acids

Carboxyl groups form amides & esters Amino groups form Schiff bases and amides Side chains show unique reactivities Cys residues can form disulfides and can be easily alkylated Few reactions are specific to a single kind of side chain
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The pathway of the ninhydrin reaction, which produces a colored product called Ruhemanns Purple that absorbs light at 570 nm. Note that the reaction involves and consumes two molecules of ninhydrin.
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Stereochemistry of Amino Acids

All but glycine are chiral L-amino acids predominate in nature D,L-nomenclature is based on D- and Lglyceraldehyde R,S-nomenclature system is superior, since amino acids like isoleucine and threonine (with two chiral centers) can be named unambiguously
The configuration of the common L-amino acids can be related to the configuration of L(-)glyceraldehyde as shown. These drawings are known as Fischer projections. The horizontal lines of the Fischer projections are meant to indicate bonds coming out of the page from the central carbon, and vertical lines represent bonds extending behind the page from the central carbon atom.
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Enantiomeric molecules based on a chiral carbon atom. Enantiomers are nonsuperimposable mirror images of each other.
Spectroscopic Properties
All amino acids absorb at infrared wavelengths Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good diagnostic device for amino acids NMR spectra are characteristic of each residue in a protein, and high resolution NMR measurements can be used to elucidate three-dimensional structures of proteins
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Cation (a) and anion (b) exchange resins commonly used for biochemical separations.
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Proteins
What Is the Fundamental Structural Pattern in Proteins?
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The Peptide Bond

s usually found in the trans configuration i as partial (40%) double bond character h s about 0.133 nm long - shorter than a typical i single bond but longer than a double bond ue to the double bond character, the six atoms D of the peptide bond group are always planar! partially positive; O partially negative N
The Coplanar Nature of the Peptide Bond Six atoms of the peptide group lie in a plane!
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The angles phi and psi are shown here

The amide or peptide bond planes are joined by the tetrahedral bonds of the -carbon. The rotation parameters are and . The conformation shown corresponds to = 180 and = 180. Note that positive values of and correspond to clockwise rotation as viewed from C . Starting from 0, a rotation of 180 in the clockwise direction (+180) is equivalent to a rotation of 180 in the counterclockwise direction (-180). (Illustration: Irving Geis.
Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)
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Peptides
Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide
Protein
One or more polypeptide chains One polypeptide chain - a monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example, is a heterotetramer It has two alpha chains and two beta chains
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What Architectural Arrangements Characterize Protein Structure?

Proteins are classed according to shape and and solubility Shape - globular or fibrous The four levels of protein structure - Primary - sequence - Secondary - local structures - H-bonds - Tertiary - overall 3-dimensional shape - Quaternary - subunit organization
(a) Proteins having structural roles in cells are typically fibrous and often water insoluble. Collagen is a good example. Collagen is composed of three polypeptide chains that intertwine. (b) Soluble proteins serving metabolic functions can be characterized as compactly folded globular molecules, such as myoglobin. The folding pattern puts hydrophilic amino acid side chains on the outside and buries hydrophobic side chains in the interior, making the protein highly water soluble. (c) Membrane proteins fold so that hydrophobic amino acid side chains are exposed in their membrane-associated regions. The portions of membrane proteins extending into or exposed at the aqueous environments are hydrophilic in character, like soluble proteins. Bacteriorhodopsin is a typical membrane protein; it binds the light-absorbing pigment, cis-retinal, shown here in red. (a, b, Illustration: Irving Geis. Rights owned by Howard Hughes
Biochemistry for the MED Boards Medical Institute. Not to be reproduced without permission.)
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Bovine pancreatic ribonuclease A contains 124 amino acid residues, none of which are tryptophan. Four intrachain disulfide bridges (SS) form crosslinks in this polypeptide between Cyc26 and Cys84, Cys40 and Cys95, Cys58 and Cys110, and Cys65 and Cys72. These disulfides are depicted by yellow bars.
Classes of Secondary Structure

All these are local structures that are stabilized by hydrogen bonds Alpha helix Other helices Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) Beta bulge
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The arrangement of hydrogen bonds in (a) parallel and (b) antiparallel -pleated sheets.
Biochemistry40 (Summer 2007) Chemistry for the MED Boards
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The structures of two kinds of -turns (also called tight turns or -bends) (Irving Geis)
Three different kinds of -bulge structures involving a pair of adjacent polypeptide chains. (Adapted from Richardson, J. S., 1981. Advances in Protein Chemistry 34:167339.)
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Supersecondary Structures
1. 2. 3. 4. -meander Greek key
Domain
Distinct compact units within a protein consisting of various elements of secondary structure 25 to 300 residues Combinations of secondary structures that form the core of a domain
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Tertiary structure
Disulfide bonds are not generally found in intracellular proteins but are sometimes found in extracellular protein May also hold together different subunits (i.e., contribute to quaternary structure)
Insulin A & B chains
Protein folding
dictated by primary structure Multiple intermediate steps Important driving forces:
Hydrophobic effect Hydrogen bonding Van der Waals Charge-charge
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Molecular chaperones
Increase the rate of correct folding of nascent polypeptide chains Aid in the assembly of multisubunit proteins Protect proteins from stress-induced damage (eg. Heat shock)
Chaperonin
Molecular chaperones assist protein folding
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Anfinsen experiment: Spontaneous renaturation of Ribonuclease A

Primary structure contains sufficient information to allow formation of secondary and tertiary structures
Predictive Algorithms
If the sequence holds the secrets of folding, can we figure it out? Many protein chemists have tried to predict structure based on sequence Chou-Fasman: each amino acid is assigned a "propensity" for forming helices or sheets Chou-Fasman: is only modestly successful and doesn't predict how sheets and helices arrange
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Relative frequencies of occurrence of amino acid residues in a-helices, b-sheets, and bturns in proteins of known structure. (Adapted from Bell , J. E., and Bell , E. T., 1988, Proteins
and Enzymes, Englewood Cliffs, NJ:Biochemistry for the MED Boards Prentice-Hall.)
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Prion Misfolding Diseases

Prion- proteinaceous infectious particles (spongiform encelopathies) Mad Cow Disease (cow) Creutzfeldt-Jacob disease (human) Scrapie (sheep)
Quaternary structure
Quaternary structure refers to the organization and arrangement of subunits in a protein with multiple subunits
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Quaternary structure
Can have more than two subunits Subunits are individual polypeptides
Pyruvate dehydrogenase complex: 60 subunits!
Levels of protein structure

Primary Secondary Tertiary Quaternary
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How Are Proteins Isolated and Purified from Cells?

The thousands of proteins in cells can be separated and purified on the basis of size and electrical charge Proteins tend to be least soluble at their isoelectric point Increasing ionic strength at first increases the solubility of proteins (salting-in), then decreases it (salting-out)
Determining the Sequence An Eight Step Strategy
1. If more than one polypeptide chain, separate. 2. Cleave (reduce) disulfide bridges 3. Determine composition of each chain 4. Determine N- and C-terminal residues
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Determining the Sequence An Eight Step Strategy
5. Cleave each chain into smaller fragments and determine the sequence of each chain 6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.
Determining the Sequence An Eight Step Strategy 7. Reconstruct the sequence of the protein from the sequences of overlapping fragments 8. Determine the positions of the disulfide crosslinks
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Step 1:
Separation of chains Subunit interactions depend on weak forces Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)
Step 2:
Cleavage of Disulfide bridges Performic acid oxidation Sulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate
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Step 3A:
Identify N- and C-terminal residues N-terminal analysis: Edman's reagent phenylisothiocyanate derivatives are phenylthiohydantoins or PTH derivatives
Step 3B: :
Identify N- and C-terminal residues C-terminal analysis Enzymatic analysis (carboxypeptidase) Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys Carboxypeptidase B (hog pancreas) only works on Arg and Lys
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Steps 4 and 5:
Fragmentation of the chains Enzymatic fragmentation trypsin, chymotrypsin, clostripain, staphylococcal protease Chemical fragmentation cyanogen bromide
Enzymatic Fragmentation
Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease C-side of Glu, Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer
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Reconstructing the Sequence

Compare cleavage by trypsin and staphylococcal protease on a typical peptide: Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-E
Reconstructing the Sequence

L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E F-S-G-I-T-P-K Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K
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Amino Acid Sequence Can Be Determined by Mass Spectrometry Mass spectrometry separates particles on the basis of mass-to-charge ratio Fragments of proteins can be generated in various ways MS can also separate these fragments
Do Proteins Have Chemical Groups Other Than Amino Acids?

Proteins may be "conjugated" with other chemical groups If the non-amino acid part of the protein is important to its function, it is called a prosthetic group. Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein, hemoprotein, flavoprotein.
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What Are the Many Biological Functions of Proteins?

Many proteins are enzymes Regulatory proteins control metabolism and gene expression Many DNA-binding proteins are gene-regulatory proteins Transport proteins carry substances from one place to another Storage proteins serve as reservoirs of amino acids or other nutrients
What Are the Many Biological Functions of Proteins? Movement is accomplished by contractile and motile proteins Many proteins serve a structural role Proteins of signaling pathways include scaffold proteins (adapter proteins) Other proteins have protective and exploitive functions A few proteins have exotic functions
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Death of a protein
In a typical day, a person who is in nitrogen balance will consume 100 grams of protein, break down 400 grams of bodily protein, resynthesize 400 grams of protein, and excrete/catabolize100 grams. Individual proteins exhibit tremendous variability in their metabolic lifetimes, from a few minutes to a few months.
Protein Turnover
Proteins in extracellular environments, such as digestive enzymes, polypeptide hormones, and antibodies, turn over quite rapidly, but proteins with predominantly structural roles, such as collagen of connective tissue, are much more stable.
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Protein Turnover
Ubiquitination Ubiquitin is a 76-amino acid residue heat-stable protein found in all eukaryotic cells. An ATP-dependent reaction with proteins links ubiquitin's C-terminal glycine to lysine amino groups in the target protein.
Protein Turnover
PEST sequences Virtually all short-lived proteins (i.e., half-lives less than 2 hours) contain one or more regions rich in proline, glutamate, serine, and threonine. Insertion of these sequences into long-lived proteins increases their metabolic lability.
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Protein Turnover
N-terminal amino acid residue An N-terminal protein residue of Phe, Leu, Tyr, Trp, Lys, or Arg is correlated with short metabolic lifetimes.
Enzymes
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What Characteristic Features Define Enzymes?
Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality Enzymes are the agents of metabolic function Catalytic power, specificity, regulation
Reaction profile showing large DG for glucose oxidation, free energy change of -2,870 kJ/ mol; catalysts lower DG, thereby accelerating rate.
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Enzyme Classification
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Vitamins
organic compounds essential in the diet in small amounts have little or no caloric value chemical composition is varied normally classified according to their polarity
Classification of Vitamins
Fat-soluble vitamins (nonpolar) Vitamin A Vitamin D Vitamin E Vitamin K Water-soluble vitamins (polar) Vitamin C Vitamin B Complex
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Fat-Soluble vitamins: A, D, E, K
Soluble in fatty tissues Stored in the body for long periods of time Not easily excreted Can be overconsumed (overdose)
Water-Soluble Vitamins: C and B Complex Soluble in water Excreted in the urine and pose little threat of overdose However, they must be consumed in sufficient amounts on a daily basis
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Nutritional Minerals
elements,other than C, H, N, and O, needed for good health. many are present as ions rather than as neutral atoms Major minerals (~4% of the bodys weight) Ca, P, Mg, Na, K, Cl, and S Minor minerals Fe, Cu, Zn, I, Se, Mn, F, Cr, and Mo
Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way?
Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! Urease is a good example: Catalyzed rate: 3x104/sec Uncatalyzed rate: 3x10 -10/sec Ratio is 1x1014 !
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The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has slightly alkaline pH optimum, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimum near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with pK' near 6 is essential to its activity. Might it be a histidine residue within the active site?
The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a function of temperature. The decrease in the activity above 50C is due to thermal denaturation.
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Specificity
Enzymes selectively recognize proper substrates over other molecules Enzymes produce products in very high yields - often much greater than 95% Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield
How Can Enzymes Be So Specific?

Lock and key was the first explanation for specificity Induced fit provides a more accurate description Induced fit favors formation of the transitionstate intermediate
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Enzyme-Linked Immunosorbent Assay (ELISA)
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Several terms to remember

rate or velocity rate constant rate law order of a reaction molecularity of a reaction
The Michaelis-Menten Equation

Louis Michaelis and Maud Menten's theory It assumes the formation of an enzymesubstrate complex It assumes that the ES complex is in rapid equilibrium with 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
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Understanding Km
The "kinetic activator constant" Km is a constant Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding
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Understanding Vmax
The theoretical maximal velocity Vmax 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 substrate Vmax is asymptotically approached as substrate is increased
The dual nature of the Michaelis-Menten equation

Combination of 0-order and 1st-order kinetics When S is low, the equation for rate is 1st order in S When S is high, the equation for rate is 0order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!
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The turnover number

A measure of catalytic activity kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. If the M-M model fits, k2 = kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per sec
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The catalytic efficiency

Name for kcat/Km An estimate of "how perfect" the enzyme is kcat/Km is an apparent second-order rate constant It measures how the enzyme performs when S is low The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together
Enzyme Units
IU (International Unit) amount of enzyme that catalyze the formation of 1 micromole of product in 1 minute Katal amount of enzyme that converts 1 mole of substrate to product in 1 second Specific activity enzyme unit per mg of protein
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Linear Plots of the Michaelis-Menten Equation
Lineweaver-Burk Eadie-Hofstee Hanes-Woolf Hanes-Woolf is best - why? Smaller and more consistent errors across the plot
The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope.
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A Hanes-Woolf plot of [S]/v versus [S], another straight-line rearrangement of the MichalelisBiochemistry for the MED Boards Menten equation.
Eadie Hofstee Linear Plot
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What Can Be Learned from the Inhibition of Enzyme Activity?

Enzymes may be inhibited reversibly or irreversibly Reversible inhibitors may bind at the active site or at some other site Enzymes may also be inhibited in an irreversible manner Penicillin is an irreversible suicide inhibitor
Lineweaver-Burk plot of competitive inhibition, showing lines for no I, [I], and 2[I]. Note that when [S] is infinitely large (1/[S] = 0), Vmax is the same, whether I is present of not. In the presence of I, the negative
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Structures of succinate, the substrate of succinate dehydrogenase (SDH), and malonate, the competitive inhibitor. Fumarate (the product of SDH action on succinate) is also shown.
Lineweaver-Burk plot of pure noncompetitive inhibition. Note that I does not alter Km but that it decreases Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).
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Lineweaver-Burk plot of pure uncompetitive inhibition. Note that I decreases Km and Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).
Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidase, which catalyzes an essential step in bacterial cell wall synthesis. Penicillin consists of a thiazolidine ring fused to a b-lactam ring to which a variable group R is attached. A reactive peptide bond in the b-lactam ring covalently attaches to a serine residue in the active site of the glycopeptide transpeptidase. (The conformation of penicillin around its reactive peptide bond resembles the transition state of the normal glycoprotein peptidase substrate.) The penicilloyl-enzyme complex is catalytically inactive. The bond between the enzyme and penicillin is indefinitely stable; that is, penicillin binding is irreversible.
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Are All Enzymes Proteins?

Relatively new discoveries Ribozymes - segments of RNA that display enzyme activity in the absence of protein Examples: RNase P and peptidyl transferase Abzymes - antibodies raised to bind the transition state of a reaction of interest
What Are the Mechanisms of EnzymeInduced Rated Accelerations? Mechanisms of catalysis:
Entropy loss in ES formation Destabilization of ES Covalent catalysis General acid/base catalysis Metal ion catalysis Proximity and orientation
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What Factors Influence Enzymatic Activity?

Six points: Rate slows as product accumulates Rate depends on substrate availability Genetic controls - induction and repression Enzymes can be modified covalently Allosteric effectors may be important Zymogens, isozymes and modulator proteins may play a role
Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes (protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible enzyme represents an -OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group.
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Proinsulin is an 86residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges.
The proteolytic activation of chymotrypsinogen. Biochemistry for the MED Boards
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The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis. It needs LDH to regenerate NAD+ from NADH so glycolysis can continue. The lactate produced is released into the blood. The muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate to fuel the citric acid cycle to obtain energy. The the MED Boards Biochemistry for heart LDH isozyme (B4) is inhibited by excess pyruvate so the fuel wont be wasted.
Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2 tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8 M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are enzymatically active as monomers.
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Carbohydrates
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D-Fructose and L-fructose, an enantiomeric pair. Note that changing the configuration only at C5 would change D-fructose to L-sorbose.
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the sweetest of all sugars (more than 50% sweeter than table sugar)
Which structure is a Haworth projection of I? Which structure is a carbon-2 (position 2) epimer of I? Which structure(s) has/have the "beta" configuration at the anomeric carbon? Which structure(s) is/are a ketose? Which structure is a pentose?
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D-Glucose can cyclize in two ways, forming either furanose or pyranose structures.
(a) Chair and boat conformations of a pyranose sugar. (b) Two possible chair conformations of -D-glucose.
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Monosaccharide Derivatives
Reducing sugars: sugars with free anomeric carbons - they will reduce oxidizing agents, such as peroxide, ferricyanide and some metals (Cu and Ag) These redox reactions convert the sugar to a sugar acid Glucose is a reducing sugar - so these reactions are the basis for diagnostic tests for blood sugar
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Structures of some sugar alcohols.

Several deoxy sugars and ouabain, which contains -L-rhamnose (Rha). Hydrogen Biochemistry for atoms highlighted in red are deoxy positions.the MED Boards
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Several sugar esters important in metabolism.

Structures of D-glucosamine and D-galactosamine.

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Structures of muramic acid and neuraminic acid and several depictions of sialic acid.
Acetals and ketals can be formed from hemiacetals and hemiketals, respectively.
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What is the Structure and Chemistry of Oligosaccharides?
Be able to identify anomeric carbons and reducing and nonreducing ends Sucrose is NOT a reducing sugar Note carefully the nomenclature of links. Be able to recognize alpha(1,4), beta (1,4), etc
Soy milk is good substitute for lactose intolerance
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The structures of several important disaccharides. Note that the notation -HOH means that the configuration can be either or . If the -OH group is above the ring, the configuration is termed . The configuration is if the OH group is below the ring as shown. Also note that sucrose has no free anomeric carbon atoms.
The structures of some interesting oligosaccharides.

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What is the Structure and Chemistry of Polysaccharides?

Functions: storage, structure, recognition Nomenclature: homopolysaccharide vs. heteropolysaccharide Starch and glycogen are storage molecules Chitin and cellulose are structural molecules Cell surface polysaccharides are recognition molecules
Starch
A plant storage polysaccharide Two forms: amylose and amylopectin Most starch is 10-30% amylose and 70-90% amylopectin Branches in amylopectin every 12-30 residues Amylose has alpha(1,4) links, one reducing end
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Amylose and amylopectin are the two forms of starch. Note that the linear linkages are (1 4), but the branches in amylopectin are (1 6). Branches in polysaccharides can involve any of the hydroxyl groups on the monosaccharide components. Amylopectin is a highly branched structure, with branches occurring every 12 to 30 residues.
Starch
A plant storage polysaccharide Amylose is poorly soluble in water, but forms micellar suspensions In these suspensions, amylose is helical iodine fits into the helices to produce a blue color
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Glycogen
The glucose storage device in animals Glycogen constitutes up to 10% of liver mass and 1-2% of muscle mass Glycogen is stored energy for the organism Only difference from starch: number of branches Alpha(1,6) branches every 8-12 residues Like amylopectin, glycogen gives a red-violet color with iodine
Structural Polysaccharides
Composition similar to storage polysaccharides, but small structural differences greatly influence properties Cellulose is the most abundant natural polymer on earth Cellulose is the principal strength and support of trees and plants Cellulose can also be soft and fuzzy - in cotton
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(a) Amylose, composed exclusively of the relatively bent (14) linkages, prefers to adopt a helical conformation, whereas (b) cellulose, with (14)-glycosidic linkages, can adopt a fully extended conformation with alternating 180 flips of the glucose units. The hydrogen bonding inherent in such extended structures is responsible for the great strength of tree trunks and other cellulose-based materials.
Structural Polysaccharides
Composition similar to storage polysaccharides, but small structural differences greatly influence properties Beta(1,4) linkages make all the difference! Strands of cellulose form extended ribbons
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The structure of cellulose, showing the hydrogen bonds (blue) between the sheets, which strengthen the structure. Intrachain hydrogen bonds are in red and interchain hydrogen bonds are in green.
Other Structural Polysaccharides

Chitin - exoskeletons of crustaceans, insects and spiders, and cell walls of fungi similar to cellulose, but C-2s are N-acetyl cellulose strands are parallel, chitins can be parallel or antiparallel
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Like cellulose, chitin, mannan, and poly(Dmannuronate) form extended ribbons and pack together efficiently, taking advantage of multiple hydrogen bonds.
Glycosaminoglycans are formed from repeating disaccharide arrays. Glycosaminoglycans are components of the proteoglycans.
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Characteristics of GAGs
GAG Localization Comments
Hyaluronate Chondroitin sulfate Heparan sulfate
synovial fluid, vitreous humor, ECM of loose connective tissue cartilage, bone, heart valves basement membranes, components of cell surfaces component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin skin, blood vessels, heart valves cornea, bone, cartilage aggregated with chondroitin sulfates
large polymers, shock absorbing most abundant GAG contains higher acetylated glucosamine than heparin more sulfated than heparan sulfates
Heparin
Dermatan sulfate Keratan sulfate
What Are Glycoproteins, and How Do They Function in Cells?

Many structures and functions! May be N-linked or O-linked N-linked saccharides are attached via the amide nitrogens of asparagine residues O-linked saccharides are attached to hydroxyl groups of serine, threonine or hydroxylysine
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The carbohydrate moieties of glycoproteins may be linked to the protein via (a) serine or threonine residues (in the O-linked saccharides) or (b) asparagine residues (in the N-linked saccharides). (c) N-Linked glycoproteins are of three types: high mannose, complex, and hybrid, the latter of which combines structures found in the high mannose and complex saccharides.
Blood Group Antigens
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Blood Group Antigens
Lipids
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Classes of Lipids
All biological lipids are amphipathic Fatty acids Triacylglycerols Glycerophospholipids Sphingolipids Waxes Isoprene-based lipids (including steroids)
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The structures of some typical fatty acids. Note that most natural fatty acids contain an even number of carbon atoms and that the for the MED Boards nearly always cis and Biochemistry double bonds are rarely conjugated.
Triacylglycerols
Also called triglycerides A major energy source for many organisms Why? Most reduced form of carbon in nature No solvation needed Efficient packing
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Triacylglycerols are formed from glycerol and fatty acids.

Triacylglycerols - II
Other advantages accrue to users of triacylglycerols nsulation I nergy without nitrogen E etabolic water M
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Glycerophospholipids
Glycerophospholipids are phospholipids but not necessarily vice versa
Phosphatidic acid, the parent compound for glycerophospholipids.

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Structures of several glycerophospholipids and space-filling models of phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol.
Ether Glycerophospholipids
An ether instead of an acyl group at C-1 lasmalogens are ether glycerophospholipids in P which the alkyl chain is unsaturated
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A 1-alkyl 2-acyl-phosphatidylethanolamine (an ether glycerophospholipid).

Ether Glycerophospholipids
latelet activating factor (PAF) is an ether P glycerophospholipid AF is a potent biochemical signal molecule P ote the short (acetate) fatty acyl chain at the N C-2 position in PAF
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The structure of 1-alkyl 2-acetyl-phosphatidylcholine, also known as platelet activating factor or PAF.
The structure and a spacefilling model of a choline plasmalogen.
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Sphingolipids
Base structure is sphingosine phingosine is an 18-carbon amino alcohol S eramides are amide linkages of fatty acids to C the nitrogen of sphingosine lycosphingolipids are ceramides with one or G more sugars in beta-glycosidic linkage at the 1hydroxyl group
Formation of an amide linkage between a fatty acid and sphingosine produces a Biochemistry for the MED Boards ceramide.
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Sphingolipids
Glycosphingolipids with one sugar are cerebrosides Gangliosides - ceramides with 3 or more sugars, one of which is a sialic acid
A structure and a spacefilling model of a choline sphingomyelin formed from stearic acid.
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The structure of a cerebroside. Note the sphingosine backbone.
The structures of several important gangliosides. Also shown is a space-filling model of Biochemistry for the MED Boards ganglioside GM1.
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Waxes
Esters of long-chain alcohols with long-chain fatty acids Highly insoluble Animal skin and fur are wax-coated Leaves of many plants Bird feathers
Figure 8.15 An example of a wax. Oleoyl alcohol is esterified to stearic acid in this case.
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Terpenes
Based on the isoprene structure Know nomenclature Understand linkage modes All sterols (including cholesterol) are terpene-based molecules Steroid hormones are terpene-based
The structure of isoprene (2-methyl-1,3-butadiene) and the structure of head-to-tail and tail-to-tail linkages. Isoprene itself can be formed by distillation of natural rubber, a linear head-to-tail polymer of isoprene units.
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Many monoterpenes are readily recognized by their characteristic flavors or odors (limonene in lemons; citronellal in roses, geraniums, and some perfumes; pinene in turpentine; and menthol from peppermint, used in cough drops and nasal inhalers). The diterpenes, which are C20 terpenes, include retinal (the essential light-absorbing pigment in rhodopsin, the photoreceptor protein of the eye), phytol (a constituent of chlorophyll), and the gibberellins (potent plant hormones). The triterpene lanosterol is a constituent of wool Biochemistry for the MED Boards fat. Lycopene is a carotenoid found in ripe fruit, especially tomatoes.
Dolichol phosphate is an initiation point for the synthesis of carbohydrate polymers in animals. The analogous alcohol in bacterial systems, undecaprenol, also known as bactoprenol, consists of 11 isoprene units. Undecaprenyl phosphate delivers sugars from the cytoplasm for the synthesis of cell wall components such as peptidoglycans, lipopolysaccharides, and glycoproteins. Polyprenyl compounds also serve as the side chains of vitamin the the ubiquinones, plastoquinones, and Biochemistry for K, MED Boards tocopherols (such as vitamin E).
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Steroids
Based on a core structure consisting of three 6-membered rings and one 5-membered ring, all fused together Cholesterol is the most common steroid in animals and precursor for all other steroids in animals Steroid hormones serve many functions in animals - including salt balance, metabolic function and sexual function
The structure of cholesterol, shown with steroid ring designations and carbon numbering.
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The structures of several important sterols derived from cholesterol.

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The Macronutrients and the Energy They Provide to the Body Fats and Oils Carbohydrates
1 Cal = 1000 cal
Membranes and Membrane Transport
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What Are the Chemical and Physical Properties of Membranes?

Structures with many cell functions Barrier to toxic molecules Help accumulate nutrients Carry out energy transduction Facilitate cell motion Assist in reproduction Modulate signal transduction Mediate cell-cell interactions
Several spontaneously formed lipid structures.

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Lipids Form Ordered Structures Spontaneously in Water

Hydrophobic interactions all! Lipid bilayers can form in several ways unilamellar vesicles (liposomes) multilamellar vesicles (Alex Bangham)
Drawings of (a) a bilayer, (b) a unilamellar vesicle, (c) a multilamellar vesicle, and (d) an electron micrograph of a multilamellar Golgi structure (X94,000).
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The Fluid Mosaic Model Describes Membrane Dynamics

S. J. Singer and G. L. Nicolson The phospholipid bilayer is a fluid matrix The bilayer is a two-dimensional solvent Lipids and proteins can undergo rotational and lateral movement Two classes of proteins: peripheral proteins (extrinsic proteins) integral proteins (intrinsic proteins)
The fluid mosaic model of membrane structure proposed by S. J. Singer and G. L. Nicolson. In this model, the lipids and proteins are assumed to be mobile, so that they can move rapidly and laterally in the plane of the membrane. Transverse motion may also occur, but it is much Biochemistry for the MED Boards slower.
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Phospholipids are arranged asymmetrically in most membranes, including the human erythrocyte membrane, as shown here. Values are mole percentages. (After Rothman and Lenard, 1977. Science 194:1744.) Biochemistry for the MED Boards
Phospholipids can be flipped across a bilayer membrane by the action of flippase proteins. When, by normal diffusion through the bilayer, the lipid encounters a flippase, it can be moved quickly to the other face of the bilayer. Biochemistry for the MED Boards
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Membranes Undergo Phase Transitions

The "melting" of membrane lipids elow a certain transition temperature, B membrane lipids are rigid and tightly packed bove the transition temperature, lipids are A more flexible and mobile he transition temperature is characteristic of T the lipids in the membrane nly pure lipid systems give sharp, well-defined O transition temperatures
An illustration of the gel-to-liquid crystalline phase transition, which occurs when a membrane is warmed through the transition temperature, Tm. Notice that the surface area must increase and the thickness must decrease as the membrane goes through a phase transition. The mobility of the lipid chains increases dramatically.
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What is Passive Diffusion?

No special proteins needed Transported species simply moves down its concentration gradient - from high [c] to low [c]
Passive diffusion of an uncharged species across a membrane depends only on the concentrations (C1 and C2) on the two sides of the membrane.
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The passive diffusion of a charged species across a membrane depends upon the concentration and also on the charge of the particle, Z, and the electrical potential difference across the membrane, Dy.
How Does Facilitated Diffusion Occur?

G negative, but proteins assist Solutes only move in the thermodynamically favored direction But proteins may "facilitate" transport, increasing the rates of transport Two important distinguishing features: solute flows only in the favored direction transport displays saturation kinetics
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Passive diffusion and facilitated diffusion may be distinguished graphically. The plots for facilitated diffusion are similar to plots of enzyme-catalyzed processes (Chapter 13) and they display saturation behavior.
How Does Energy Input Drive Active Transport Processes?

Energy input drives transport Some transport must occur such that solutes flow against thermodynamic potential Energy input drives transport Energy source and transport machinery are "coupled" Energy source may be ATP, light or a concentration gradient
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The Sodium Pump

aka Na,K-ATPase Large protein - 120 kD and 35 kD subunits Maintains intracellular Na low and K high Crucial for all organs, but especially for neural tissue and the brain ATP hydrolysis drives Na out and K in Alpha subunit has ten transmembrane helices with large cytoplasmic domain
(a) A schematic diagram of the Na+,K+-ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na+ ions are transported out of the cell, and K+ ions are transported in. The transport stoichiometry is 3 Na+ out and 2 K+ in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na+,K+-ATPase by binding on the extracellular surface of the pump protein. (b) Stereo views of the Na+,K+-ATPase.(From Biochemistry for the MED Boards Hkansson,K.O., 2003. The crystallographic structure of Na,KATPase N-domain of 2.6 resolution. J Mol Biol 332:1175-1182.)
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6/13/11

On Life and Chemistry..

Scope of this Review

Main source of energy in most organisms

The language of nucleic acids

What makes us human

chromosomes, arranged in 23 pairs, in each cell in the body

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Distinctive Properties of Living Systems

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Methods for Separating and Purifying Biomolecules

Methods for Determining Biomolecular Structures

Preparations for Studying Biochemical Processes

Major Causes of Diseases

Major Causes of Diseases

Amino Acids Building Blocks of Proteins

20 Common Amino Acids

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

pKa Values of the Amino Acids

pKa Values of the Amino Acids

pKa Values of the Amino Acids

Biochemistry for the MED Boards

Titration of glutamic acid.

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Reactions of Amino Acids

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Stereochemistry of Amino Acids

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry for the MED Boards

What Is the Fundamental Structural Pattern in Proteins?

The Peptide Bond

Biochemistry for the MED Boards

The angles phi and psi are shown here

What Architectural Arrangements Characterize Protein Structure?

Classes of Secondary Structure

Biochemistry for the MED Boards

Biochemistry for the MED Boards

Biochemistry40 (Summer 2007) Chemistry for the MED Boards

Biochemistry for the MED Boards

Insulin A & B chains

Molecular chaperones assist protein folding

Anfinsen experiment: Spontaneous renaturation of Ribonuclease A

Biochemistry for the MED Boards

Prion Misfolding Diseases

Pyruvate dehydrogenase complex: 60 subunits!

Levels of protein structure

How Are Proteins Isolated and Purified from Cells?

Determining the Sequence An Eight Step Strategy

Determining the Sequence An Eight Step Strategy