Exam 2 Study Guide (Max Sokolov) PDF

Lecture 13: Catalytic Mechanisms and Enzyme Regulation Transition State -Reaction occurs between electron-deficient atom (electrophiles) and electron-rich atom (nucleophiles) -Chemical bond is formed when a nucleophile donates an electron pair to an electrophile - Ex.) the pi bond electrons of the d...

Lecture 13: Catalytic Mechanisms and Enzyme Regulation Transition State -Reaction occurs between electron-deficient atom (electrophiles) and electron-rich atom (nucleophiles) -Chemical bond is formed when a nucleophile donates an electron pair to an electrophile - Ex.) the pi bond electrons of the double bond are rxting with hydronium Enzymes stabilize transition states -Short living intermediates between reactant and product with energy maxima are termed transition states -Enzymes bind transition states with a high affinity which stabilizes them -Stabilizing transition state reduces its energy, which means that less energy is required to attain it The activation energy ∆E╪ becomes reduced and the rate constant k increase i.e. the reaction proceeds faster Catalytic mechanisms -The active (catalytic) sites of the enzymes has the structure which is uniquely suited to lower ∆G╪ of the reaction. The following underlying mechanisms are recognized: Proximity and strain effects Electrostatic effects Acid-base catalysis Covalent catalysis Proximity and Strain Effects - Substrate fits the catalytic site with optimal orientation between enzyme and substrate functional groups -Enzyme conformation changes to give a strained ES complex Electrostatic Effects - Substrate binding site excludes H2O This lowers the dielectric constant, thus strengthening electrostatic interaction between E and S Acid-base Catalysis - Acid-base catalysis (proton transfer) is important factor in chemical rxns Ex.) Hydrolysis of esters can catalyze by free hydroxide ion (HO-) General Base Catalysis - Enzymes conducts general base catalysis, when an amino acid in its active site accepts H+ from the substrate General Acid Catalysis - Enzyme conducts general acid catalysis, when an amino acid in its active site donates H+ to the substrate. Acid-base catalysis by enzymes - Functional groups in the side-chain of certain amino acids can act as either a H+ donor (general acid) or H+ acceptor (general base) Histidine can act as either a general acid or base Mechanism of Triose Phosphate Isomerase - A glycolytic enzyme which catalyzes isomerization of DHAP to GAP -The rxn involves formation of two enediolate transition states (TS) flanking enediol intermediate 2-Phosphoglycolytic acid inhibits triose phosphate isomerase - 2-phosphoglycolic acid (PGA) resembles enediolate 1 Inhibits the enzyme as a transition state analog Covalent Catalysis - a nucleophile side chains forms an unstable (transient) covalent bond to the substrate Exemplified by polypeptide digestion by the serine proteases Oxygen or serine attacks the carbonyl group of peptide An ester bond is formed and the peptide bond is broken The acyl-E intermediate is then hydrolyzed by water - Polypeptide digestion by serine protease Mechanism of Chymotrypsin - breaks peptide bond next to large nonpolar amino acid (Phe, Tyr, Trp) 1.) Ser^195 acts in concert with His^57 and Asp ^102 as a so-called protease triad (Asp-His-Ser) 2.) The carboxyl group of Asp^102 polarizes imidazole ring of His^57 thus allowing it to act as a general base (take an H+) 3.) Activated His^57 removes H+ from the hydroxyl group of Ser^195, and by doing so makes it a better nucleophile 4.) Activated Ser^195 attacks the peptide bond DFP inhibits chymotrypsin - All serine proteases are inhibited by diisopropylfluorophosphate (DFP) One 1 Ser in the active site of the enzyme becomes modified by DFP In chymotrypsin, Ser^195 becomes labeled by DFP The roles of amino acids in enzyme catalysis - Of the 20 AAs found in proteins only those with polar and charged side-chains actually participate in catalysis Serine, threonine, and tyrosine (hydroxyl) Cysteine (thiol) Glutamine and asparagine (amide) Glutamate and aspartate (carboxylate) Lysine (amine) Arginine (guanidinium) Histidine (imidazole) The roles of metal ions in enzyme catalysis - Alkali and alkaline Earth metals: (Na+, K+, Mg++, Ca++) loosely bound, structural role -Transition Metals: (Zn++, Fe++, Cu++) are often tightly bound and involved in catalysis Are electrophiles because they act as Lewis acids (electron pair acceptor) The roles of coenzymes in enzyme catalysis -Coenzymes can be classified according to the function into the three following group: Electron or Hydrogen Transfer: NAD+, NADP+, FAD+, FMN, CoQ, tetrahydrobiopterin (BH4) Group Transfer: thiamine pyrophosphate (TPP) - aldehyde CoA, pyridoxal phosphate - amino group; Biotin, tetrahydrofolate (TH4) and S-adenosylmethionine (SAM) - one carbon transfer High-energy transfer Potential: UDP-glucose, cytidine, diphosphate ethanolamine (CDP) Mechanism of alcohol dehydrogenase -Catalyzes reversible conversion of ethanol to acetaldehyde In humans, the forward rxn clear blood alcohol thus contributing to HANGOVER Yeasts utilize the reverse rxn to regenerate NAD+ during alcoholic fermentation Oxidation of -OH is coupled with reduction of NAD+ (involves movement of H+ and 2e-)  In the active site, Zn+ is coordinated by Cys^48, Cys^174, and His^67 Ser^48 interacts with the substrate H2O, which is a ligand of Zn++ in free enzyme, is displaced by ethanol during catalysis NAD+ accepts H+ and 2e- from ethanol and acetaldehyde is formed Mechanisms of enzyme regulation - To regulate their vast network of metabolic pathways, living organisms utilize the following: Genetic control Covalent modification Allosteric regulation Compartmentalization Genetic Control - Many enzymes are controlled genetically so their genes are only expressed in the cells that need them - Enzyme Induction: Some enzymes are synthesized by cells only than their substrates are present in the media (occurs at transcriptional level) Covalent Modification - Many enzymes are regulated by reversible covalent modification of specific residues, called regulatory sites. - Phosphorylation or attachment of phosphate group to serine, threonine or tyrosine residues is the most common type of modification (others include methylation or acetylation) -Zymogenes: enzymes produced as inactive precursors Are converted into active enzymes by the irreversible cleavage of one or more peptide bonds Allosteric Regulation - Enzyme is regulated through binding of the effector to The effector Binding site distinct from the functional (catalytic) site Binding of the effector affects the functional site, thus stimulating or suppressing the catalysis - Homotropic: the effector is the same as the substrate - Heterotropic: the effector is different from the substrate - Allosteric enzymes display sigmoidal (not hyperbolic) Michaelis-Menten plots Their activity can be altered markedly when either an activator or an inhibitor is bound to the enzyme Compartmentalization - The physical separation of competing reactions - creation of diffusion barriers confiding enzymes and substrates - specialized reaction conditions such as pH - Damage control: segregation of potentially toxic reaction products Lecture 14: Monosaccharides Monosaccharides - Water soluble, white crystalline solids that have a sweet taste - name could reflect the number of carbons: trioses (C3), tetroses (C4), pentoses (C5), hexoses (C6) - have many hydroxyl groups, and one carbonyl group, as an aldehyde or a ketone. (Aldose or ketose) Fischer and Haworth Projections - Fischer Projection: A convenient way to show linear structure of monosaccharides - Haworth Projection: the best for depicting cyclic monosaccharides Fischer Projection is not flat - Fischer projection does not accurately represent the real structure (convenient) -It implies that “black” horizontal bonds Point towards the viewers, and “striped” vertical bonds point towards the page. The simplest aldose and ketose L and D-Glyceraldehyde - Glyceraldehyde has a asymmetric carbon This makes glyceraldehyde a chiral compound, which exist as L- and D- glyceraldehyde -Chiral compounds are optically active (rotate polarized light) L- and D- rotate light in different directions Monosaccharides are Chiral Compounds -Most monosaccharides are chiral compounds (carbon atom must be connected to 4 diff. groups) There are 2^n possible stereoisomers for a monosaccharide with n chiral carbons - Epimers: mirror-image stereoisomers that are different at one chiral center Carbon Atom numeration. L and D Configuration - In the fischer projection, The carbon count starts from the end with an aldehyde or ketone - The reference for L- or D-configuration is the asymmetric carbon which is the most remote from an aldehyde/ketone Living Cells Predominantly use D-sugars - The stereoisomers of the same sugar have identical chemical properties, yet they are biochemically different due to their different shapes (configuration) - almost all natural sugars are in the D - configuration, which means that -OH of the reference carbon sticks to the right on the Fisher projection Enantiomers, Diastereomers, and epimers - Enantiomer: D- and L-Ribose are mirror image of each other - Diastereomer: D-Ribose and D-Arabinose are not mirror images - Epimer: Diastereomers with one asymmetric carbon D-Ribose and L-Arabinose Formation of Hemiacetal and Hemiketal - The aldehyde and Ketone groups react with the alcohol group - the products hemiacetal or hemiketal are unstable when the rxn occurs between two different molecules Cyclization of Monosaccharides - In a monosaccharide, an aldehyde or Ketone can react with alcohol groups from the same molecule, if it's backbone comprises four carbons or more The resulting intramolecular hemiacetal or hemiketal is stable enough to hold the sugar in a cyclic form Cyclisation of Glucose 1.) OH on C-5 connects to C-1 2.) Hemiacetal can lock C-1 in two configurations (alpha and beta) 3.) In Haworth projection of an alpha-sugar, -OH sticks down in respect to the ring 4.) Cyclic hemiacetal constantly reverts to the open configuration. Pyranoses and Furanoses - Two types of rings formed by sugar resemble organic molecules pyran and furan - Based on that, each sugar structure can be classified as pyranose or furanose What’s in the name alpha-D-Glucopyranose? - “gluco” → glucose - “pyranose” → six-joints ring which resembles pyran - “D” because in the open configuration -Oh of C-5 (now O in the ring) sticks to the right - -OH of C-1 has near equal chance to point either up or down in respect to the ring after cyclisation - This makes C-1 chiral, and the resulting alpha- and beta-isomers are called anomers (term for sugar epimers) Glucose can also be a furanose - Glucofuranose is formed when -OH on C-4 rxts with an aldehyde on C-1 Equilibrium mixture of D-Glucose -Water solution of D-Glucose contains equilibrium mixture of alpha-D-Glucopyranose (38%) Beta-D-Glucopyranose (62%) alpha-D-Glucofuranose (C-OH) Product is called sugar alcohols Oxidation of Carbonyl or Hydroxyl -Aldehyde and primary alcohol groups can be oxidized into the carboxylic group, -COOH The products are called sugar acids Cyclisation of D-Glucuronic Acid - Sugar acids forms a cyclic ester, called lactone Reducing and nonreducing sugars - A sugar is called reducing if it can be oxidized by weak oxidizing agents (e.g. Benedict’s Reagent) to reduce metal ions such as Cu++ or Ag+ to insoluble products During the rxn, the aldehyde group is oxidized to carboxyl -Thus, reducing sugar must have an open aldehyde group. All monosaccharides are reducing agents - Note: All monosaccharides are reducing sugars, because they all have interconvertible aldehyde or ketone groups when in open configuration (see migration of carbonyl) Disaccharides and polysaccharides are reducing only if they have an “unlocked” anomeric C within hemiacetal or hemiketal Migration of carbonyl underlies aldose-ketose interconversion Lecture 15: Disaccharides , Homoglycans Formation of Acetals and Ketals - Hemiacetals rxt with alcohols to form acetals - Hemiketals rxt with alcohols to form ketals Formation of Glycosides - Acetals and ketals of sugars are called glycosides - Anomeric -OH groups are being replaced with -OR - Anomeric carbon becomes “locked” in the alpha- or beta- configuration N-Glycosides - Anomeric carbon in sugars also rxt with amines to form N-glycosides Nucleosides (e.g. guanosine, adenosine, etc.) are the N-glycosides of a sugar and are a purine or a pyrimidine Disaccharides -Disaccharides are comprised of 2 monosaccharide residues 1 provides hemiacetal/hemiketal, the other provides -OH group Resulting linkage is called a glycosidic bond Glycosidic Bond - Glycosidic bond is described by a combination of 1-2 Greek letters and 2 numbers (e.g. alpha-Beta (1,4) The greek letters indicate alpha- or beta- configuration of the connected anomeric carbons. Only one letter means that the seconds carbon is not anomeric Two numbers in the parentheses indicate the carbons are connected by the glycosidic linkage. Disaccharide Maltose - Maltose, or malt sugar, is an intermediate product of starch hydrolysis Composed of 2 molecules of D-glucose connected by a alpha(1,4) glycosidic linkage In solution exists as an equilibrium mixture of alpha and beta–maltose, due to mutarotation Reducing sugar, because its glucose has a free hemiacetal group that opens to aldehyde Understanding structure of maltose Disaccharide Lactose - Lactose is a disaccharide found in milk Composed of a molecule of galactose connected by a Beta(1,4) linkage to glucose Can be in alpha or beta configuration, based on configuration of glucose component Reducing sugar, because its glucose has a free hemiacetal group that opens to the aldehyde Understanding structure of lactose Disaccharide Sucrose - Sucrose is a common table sugar Composed of a molecule of glucose connected alpha,beta (1,2) linkage to fructose Non Reducing sugar because in both residues the anomeric carbons are locked Understanding structure of sucrose Polysaccharides: Homoglycans - Polysaccharides are polymers comprised of 20 or more monosaccharide residues - classified as homoglycans if made of one type of sugar monomer, otherwise they are known as heteroglycan They are synthesized in the cell without a template, which makes them heterogeneous in terms of the length and the composition -Homoglycans (starch and glycogen): major chemical energy storage -Homoglycans (cellulose and chitin): major structural materials Starch and Glycogen - Oxidation of D-Glucose is used as the energy source by all living cells In the cells, D-Glucose is stored as homoglycan this homologan is called starch in plants and fungi, and glycogen in animals Amylose is a component of starch - Linear polymer of D-glucose residues connected by alpha-(1,4), glycosidic links Forms a left-handed helix Amylopectin is a component of starch - Amylopectin is a branched version of amylose with alpha (1,6) glycosidic links 1 branch per 25 residues, and 15-25 residues-long Breaking Starch - Hydrated (swollen) starch is a substrate of 2 glycosidases, alpha- and beta-amylases, and debranching enzyme - Alpha-amylase is a endoglycosidase that catalyzes random hydrolysis of the internal alpha (1,4) bonds of amylose and amylopectin - Beta-amylase is a exoglycosidase that catalyzes hydrolysis of terminal alpha (1,4) bonds, producing maltose Debranching enzyme breaks beta (1,6) glycosidic bond Glycogen - Glycogen is the storage of D-glucose in animals Can account to 10% of the mass of the liver, and 2% of the mass in the muscle Structurally similar to amylopectin, but branches are smaller and more frequent (every 8-12 residues) Are larger than starch molecules Cellulose and chitin - Cellulose is structural homoglycan of the rigid cell walls that surround many plant cells - Chitin is structural homoglycan found in the exoskeletons of insects, crustaceans, and also in the cell walls of most fungi and red algae Both are the most abundant organic compounds on Earth Cellulose - In cellulose, D-Glucose residues are connected via Beta (1,4) glycosidic linkage Each residue is rotated 180 degrees relative to its neighbor No branching → linear with only beta connection  - Extensive intermolecular H bonding within and between cellulose chains leads to the formation of bundles or fibrils Cellulose fibrils are insoluble in water and are quite strong and rigid Cotton fibers are 100% cellulose, wood is 50% cellulose Why humans don’t graze - Mammals cannot metabolize cellulose because they do not express Beta-glycosidases Ruminants such as cows and sheep keep microorganisms in their stomach (rumen) that breaks Beta (1,4) glycosidic linkage in dietary cellulose (grass) Chitin - Chitin is a structural component of exoskeleton of invertebrates Homoglycan of N-acetyl-Beta-D-glucosamine (GlcNAc) GlcNAc are connected via Beta (1,4) glycosidic linkage Each residue is rotates 180 degrees relative to its neighbors - Strength is due to hydrogen bonding and also non-sugar components, including protein and inorganic material Lecture 16: Heteroglycans, glycoconjugates Heteroglycans and Glycoconjugates - Heteroglycans: long polymers that are made of more than one type of monosaccharide -Glycoconjugates: heteroglycans conjugates to proteins or peptides Classified as proteoglycans, peptidoglycans and glycoproteins Proteoglycans - Found in the extracellular matrix (connective tissue) of multicellular organisms e.g. cartilage - Protein components are conjugated to heteroglycan, classified as glycosaminoglycan (GAG) This structure is immobilized on a large heteroglycan called hyaluronic acid Hyaluronic Acid - Hyaluronic acid: unbranched heteroglycan made of repeating disaccharide unit, D-glucuronate (GlcUA) and amino sugar connected by Beta(1,3) and Beta(1,4) glycosidic linkage Aminosugar is either D-N-acetylgalactosamine (GlcUA) or D-N-acetylglucosamine (GlcNac) Structure of D-Glucuronate (GlcUA) Structure of N-Acetyl-D-Galactosamine Disaccharide unit of hyaluronic acid Table 8.2 Structures of some common polysaccharides Peptidoglycans - Christian Gram invented a strain that makes some bacteria blue, called gram-positive. - In such gram-positive bacteria, the peptidoglycan cell wall is exposed and is much thicker than in gram-negative bacteria - Heteroglycan is linked to small peptides - Repeating disaccharide unit of N-acetyl-Beta-D-glucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) connected by Beta(1,4) link N-acetylmuramic Acid (MurNAc/NAM) Peptide component of peptidoglycans - The peptide component of peptidoglycan varies among bacteria - In Staphylococcus aureus, it is a tetrapeptide unit made of alternating L- and D-amino acid residues - The E-amino group of L-lysine is crosslinked to the alpha-carboxyl group of D-Alanine via pentaglycine bridge Penicillin is inhibitor of peptidoglycan biosynthesis - Antibiotic penicillin mimics D-Ala-D-Ala residues of the tetrapeptide unit present on immature peptidoglycan Irreversibly inhibits the bacterial cell wall synthesis Glycoproteins - Heteroglycan ( 4-20 residues-long oligosaccharide) is covalently linked to a protein When the acceptor is a side chain of serine , oligosaccharide is O-linked (alpha), when asparagine is -N linked (Beta) Lecture 17: Cell-Surface and nuclear receptors Biological signaling: Introduction - Metabolism, a sum of anabolism and catabolism, is coordinated by nervous and endocrine systems - Endocrine system acts by secreting signaling molecules called hormones into the blood Hormones travel through blood until they reach a target cell, and bind to their receptors, which trigger specific cellular responses Two different strategies exploit cell-surface and nuclear receptors Cell-Surface Receptors - Water soluble hormones freely travel in the blood, but cannot cross plasma membrane to enter the cell - Receptors transverse the plasma membrane. The extracellular domain tightly binds the hormone (lock and key) Hormone-bound receptor assumes “active” configuration, propagating across the plasma membrane to its intracellular domain , which triggers the response inside the cell Hormone signal is amplified by 2nd Messengers - The intracellular actions of hormones are mediated by a group of molecules referred to as 2nd messengers Act to modulate enzymes, often by an enzyme cascade Original signal creates amplified and diversified response - 2nd messengers can be water soluble: AMP and cyclic GMP, inositol-1,4,5-triphosphate, or hydrophobic (diacylglycerol) Cell-surface receptors - Ligands (hormones) are water soluble - Receptor is located in the plasma membrane G protein-coupled receptors (GPCRs) Receptor tyrosine kinase (RTKs) G Protein-coupled receptors -GPCRs is the most abundant class of cell-surface receptors activated by a multitude of ligands - Each GPCR “communicates” through dedicated “ translator”, heterotrimeric G protein Heterotrimeric due to 3 diff. Subunits: G-alpha, G-beta, G-Upsilon “G protein” because G-alpha binds and hydrolyzes GTP (GTP → GDP + Pi) Hydrolysis of bound GTP serves as the “off” switch for signal transduction Activation Cycle of heterotrimeric G proteins 1.) Activated receptor makes G-alpha,beta,upsilon (a) to replace Ga-bound GDP with GTP (b) to dissociate into free G-alpha and G-beta,upsilon propagating signal 2.) Inactive G-alpha,beta,upsilon re-assembles. Then G-alpha hydrolyzes GTP. Signal propagation stops Glucagon receptor controls release of glucose from glycogen - Pancreatic hormone glucagon binds to its receptor Glucagon signal is mediated by heterotrimeric G protein, called Gs - Glucagon-bound receptor activates Gs, which releases GTP-bound G-alpha(s) - GTP-bound G-alpha(s) activates adenylate cyclase, generating a spike of cAMP which activates cAMP-dependent protein kinase PKA PKA phosphorylates phosphorylase kinase, which in turn activates glycogen phosphorylase PKA translocates to the nucleus and phosphorylates the cAMP response element binding protein, CREB, a transcription factor -G-alpha(s) turns off after it hydrolyzes bound GTP and re-associates with G-beta,upsilon cAMP is cleared by phosphodiesterase Structure of the 2nd messenger molecule cAMP Receptor Tyrosine kinase, RTK - receptor “communicates” through the tyrosine kinase activity of its intracellular domain, unleashed in response to the ligand (hormone) binding to the extracellular domain - Self-phosphorylate first, and then begins to phosphorylate other proteins Insulin receptor controls glucose uptake - Forms alpha2 beta2 tetramer in the plasma membrane The alpha subunits organize insulin-binding site The beta subunits display tyrosine kinase activity “communication” 1.) Insulin binds to the insulin receptor in the insulin-responsive tissues (muscles and adipose tissue) 2.) Triggers translocation of Glut-4 to plasma membrane which increases glucose uptake and metabolism 3.) Influx of glucose 4.) Glycogen synthesis 5.) Glycolysis (pyruvate) 6.) fatty acid synthesis Nuclear receptors of steroid hormones - Steroid hormone is a steroid that acts as a hormone. Helps control metabolism, inflammation, immune function, salt and water balance, and development of sex characteristics Corticosteroids are produced in the adrenal cortex Sex steroids are produced by the gonads or placenta -Based on the receptor to which they bind, steroid hormones are subdivided into glucosteroids, mineralocorticoids, androgens, estrogens, and progestogens Steroid hormones Production - Cholesterol is a precursor in the biosynthesis of steroid hormones - Cholesterol is a C-27 lipid, which is formed from the linear triterpene squalene (C-30) by intramolecular ring closure Structure of Animal Steroids Steroid Hormones Action - Steroid hormones are hydrophobic and insoluble in water. They travel in the blood as a complex with carrier proteins via hormone-binding globulins. Cross the plasma membrane due to their hydrophobicity In the cytoplasm, steroid hormones may or may not undergo enzyme-mediated modification (reduction, hydroxylation, aromatization) They then then bind to steroid hormone receptors that are DNA-binding proteins located in cytoplasm or in the nucleus In the absence of the hormone, steroid hormone receptors stay in a complex with heat-shock protein 90 chaperone Nuclear Receptors of thyroid hormones - Tyrosine based hormones that contain iodine -regulate every aspect of metabolism in most cells - produced by the thyroid gland Thyroid Hormones Action - Travel in the blood attached to a carrier protein, thyroxine-binding globulin - Only free thyroxine and T3 can enter the target cell, and thyroxine is converted to T3 Receptor for T3 is located in the nucleus - Receptor bind DNA as a heterodimer with 9-retinoic acid