lec07.docx

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Department
Biological Sciences
Course
BIOC12H3
Professor
Shelley A.Brunt
Semester
Fall

Description
lec07 collagen / myoglobin /hemoglobin / immunoglobulins + enzyme properties 1. collagen with unique fibrils having unique role of h bonds 2. different kinds of interactions that are critical for tertiary and quaternary structure 3. classifications of enzymes 4. cofactors and enzymes 5. midterm july 12 must relate structure to function 1. talk about a. collagen i. covalent interaction 1. critical 2. important for strength and flexibility b. myoglobin and hemoglobin i. bind molecular oxygen through heme (prosthetic groups) ii. important for binding oxygen iii. their binding are not much different form how enzymes interact c. antibodies i. specific binding interactions with antigens ii. antibodies are specific 1. binds to specific antigens 2. specific binding interactions and critical domains that interact with the antigen 3. FC region interacts with phagocytes to destroy antigens  verify 2. structures and how proteins fold to result in particular function a. structure affected by function collagen 1. a fibrous protein different from alpha keratin 2. human type III collagen has three identical extended left hand helices a. collagen made up of 3 identical left handed helices (LEFT) b. these coil around each other to form a right handed SUPER coil c. triple helices aggregate in staggered association to form strong insoluble fibers d. considered extended because it is 3 amino acids more per turn than a classic alpha helix e. covalent cross links between molecules and within strand gives it its strength 3. major component of connective tissues of vertebrates a. stabilized by INTERCHAIN h bonds i. interchain vs intrachain? 1. See page 3 b. the helical region consists of multiple repeats of the sequence -Gly-X-Y- (X often a protein; Y often a modified protein called 4-hydroxyproline) c. glycine is in the central axis of the triple helix i. where the tight packing of the three protein strands exclude all other amino acids d. for each Gly-X-Y triplet, one h bond forms between i. the amide hydrogen atom of the glycine in one chain ii. with the carbonyl oxygen atom of residue X in an adjacent strand 1. visualize this e. h bonds involving hydroxyl group of hydroxyproline may also contribute to stabilization f. there are NO INTRAchain h bonds which are often found in an alpha helix i. no alpha helices in collagen due to the limited flexibility of proline and hydroxyproline 1. no alpha helices in collagen due to presence of proline ii. glycine is unusual in alpha helices because its flexibility tends to disrupt the structure 1. however, it is the presence of glycine at every 3rd position that allows the tightly wound left handed helix to accomodate the proline g. importance of glycine and proline i. why is proline present? 1. hydroxylation of proline is done in presence of vitamin C a. lack of vitamin C results in scurvy tropocollagen triple helices 1. main point of slide 5? 2. tropocollagen triple helices align side by side a. looks like a braid b. aligned left to right from decreasing to increasing order conformation of a single strand of the collagen triple helix 1. Glycine-Proline-Hydroxyproline 2. Gly-X-Y a. visualize the triple helix b. slide 6 collagen strands 1. each strand is h bonded to the other two strands a. every 3rd residue is glycine 2. hydroxylation is a type of post translational modification that is critical for a number of proteins a. 4-hydroxyproline i. formation is catalyzed by an enzyme that carries out hydroxylation of proline after protein synthesis 1. adds a hydroxyl group to carbon 4 of proline interchain h bonds in collagen 1. inter  between two amino acids a. h bonds form between amide H on glycine and carbonyl oxygen of proline 2. intra  within an amino acid collagen also contains a 5-hydroxylysine 1. 5-hydroxylysine sometimes linked to carbohydrate a. i.e. collagen is a glycoprotein via o-linked glycosylation i. which normally occurs via serine 1. what does serine do? a. undergoes phosphorylation ii. what is a glycoprotein? 1. Proteins that contain oligosaccharide chains covalently attached to polypeptide side chains 2. Often important integral membrane proteins a. Play a role in cell-cell interactions iii. what is o-linked glycosylation? 1. Is the attachment of a sugar molecule to an oxygen atom in an amino acid residue in a protein 2. Form of glycosylation that occurs in golgi apparatus of eukaryotes and bacteria 3. through OH groups 2. second modifcation that collagen undergoes a. contains a 5-hydroxylysine that is linked to carbohydrates i. glycine does not normally have an OH group 1. but when it does, it is hydroxylated to help ensure collagen has its strength in bonds a. as a result of o-linked glycosylation b. there are also o-linked glycoproteins in the nucleus i. which suggest additional roles 1. ... in what? in glycoproteins? covalent cross links in collagen 1. the CH2-NH3+ groups on the side chains of some lysines and hydroxylysines are converted enzymatically to aldehyde groups (-CHO) a. produces allysine i. can react with side chains of lysine (hydroxylysine) to form schiff bases 1. schiff bases a. a covalent cross link between collagen molecules i. fibrils make up the collagen cables 2. main point a. allysine is formed through enzymatic conversion of NH amine group to CHO aldehyde group b. allysine and lysine interact to form a schiff base c. schiff base formation critical in stabilizing the molecule i. via intramolecular interactions d. functional component of collagen is the individual fibrils that come together i. structural capacity 3. two allysine residues can also condense in aldol condensation reactions to form cross-links between individual strands of the triple helix a. via intramolecular binding b. cross links between individual strands of the triple helix i. what are cross-links? 1. A bond that links one polymer chain to another a. Either covalent or ionic bonds 4. hydroxylation of collagen is impaired in the absence of vitamin C a. humans cannot make vitamin C i. need external source of vitamin C b. without vitamins, we don't get co-factors i. no cofactors  enzymes don't work 5. three main components important in collagen a. hydroxylation of proline b. acetylation of lysine c. hydroxylation of lysine 6. role of hydroxylation in stability of collagen a. without h bonds between hydroxyproline residues which assist stabilization i. the collagen helix is unstable ii. and loses most of its helical content at temperatures above 20 degrees 1. such collagens are formed by experimental animals in the absence of ascorbic acid (vitamin C) a. normal collagen is more stable and resists thermal denaturation until a temperature of about 40 degrees i. functional temperature ~37 degrees b. might be a repeat of information i. what would happen to the stability of the collagen molecule without a hydroxylated proline? 1. the collagen helix is unstable and loses most of its helical content ii. stability of collagen resists thermal denaturation up to 40 degrees 1. without this stability, thermal denaturation occurs at 20 degrees iii. vitamins serve as cofactors for enzymes 1. implicated in substrate binding sites denaturation of collagen containing a normal content of hydroxyproline vs abnormal content of hydroxyproline (i.e. no hydroxyproline content) 1. denaturation curve in the presence and absence of hydroxyproline a. up to 40 degrees, normal collagen persists b. up to 20 degrees, abnormal collagen already scarce i. becomes hypothermic 1. verify hypothermic vs hyperthermic meaning a. hypothermic i. body temperature dangerously low b. hyperthermic i. body temperature dangerously high collagen highly post-translationally modified 1. pre-pro-collagen molecules being made  appropriate hydroxylations taking place in ER 2. collagen pathway a. translation on ribosome b. hydroxylation of proline and lysine c. release form ribosome and addition of ER sugars d. formation of triple helix and folding of globular domains e. secretion from cell f. removal of N and C terminal domains g. de-amination of lysine residues to form allysine (aldehyde) and resulting formation of cross-links i. allysine important in formation of cross links myoglobin and hemoglobin (bind molecular oxygen) through heme (prosthetic) groups 1. prosthetic group a. a protein bound organic molecule essential for the activity of a protein i. e.g. heme 1. porphyrin ring structure complexed to iron (Fe) 2. found in myoglobin and hemoglobin 2. blood test for presence of iron a. diagnostic of how well you can carry oxygen myoglobin 3. myoglobin for a single unit oxygen a. consists of 8 alpha helices b. the heme prosthetic group binds oxygen c. histidine 64 forms a h bond with oxygen d. histidine 93 is complexed to the iron atom of the heme e. oxygen is not directly binding to protein i. it is binding to the heme prosthetic group 4. role of two histidines a. His64 b. His93 c. what is the characteristic of histidine? i. it is a basic amino acid ii. pI of approximately 7 iii. very good acid / base acceptor at neutral pH 1. therefore one of the most common amino acids at active sites human hemoglobin 1. composed of two alpha subunits and two beta subunits 2. each subunit with a heme prosthetic group that transports oxygen in red blood cells 3. binding and release of oxygen is regulated by allosteric interactions a. i.e. resembles an enzyme i. binds an effect that modulates the activity of hemoglobin b. what are allosteric interactions? i. requires assistance of some other molecules to alter the active site of protein ii. allosteric regulation 1. regulation of enzyme or other protein by binding an effector molecule at protein’s allosteric site a. allosteric site is site other than the protein’s active site i. allosteric sites are physically distinct from active site 2. effectors that enhance activity are activators 3. effectors that decrease activity are inhibitors 4. allosteric regulation are examples of control loops a. feedback from downstream products b. feedforward from upstream substrates 4. haemoglobin has 4 heme groups a. binds more oxygen than myoglobin found in muscles b. oxygen binding and release done through allosteric interactions (related to enzymes) i. requires assistance of some other molecules to alter the active site of protein tertiary structure of myoglobin 1. alpha globin and beta globin are super imposable a. form equals 2. function a. reversible binding of oxygen heme interaction with histidine 1. what is the role of histidine? a. way in which heme interacts with histidine results in conformational changes in the binding of oxygen conformational change in hemoglobin 2. induced by oxygenation a. when oxygenated, the proximal histidine is pulled toward the porphyrin ring of the heme i. the helix also shifts during the T to R transition 1. localized change in protein conformation based on bound oxygen 3. what happens when oxygenation is induced? a. i.e. when oxygen binds i. the proximal histidine (which is attached to heme group) is pulled forward 1. slight change in conformation of that specific active binding site a. binding of ligand alters the cleft or active site that is critical for function i. porphyrin ring is pulled towards the heme just enough to change that region of the active site 1. switches from T to R transition a. enzymes; T for tense, R for relaxed b. relaxed state allows it to function better c. ability to go from T (less efficient) to a more R form (more sufficient for function) enzymes 1. concept of enzymes 2. reflect using what you know a. why are enzymes important? i. important in studying diseases, active sites are important in drug rehabilitation b. why is it important to understand the structure of active sites? i. ??? 3. biological catalysts a. protein that i. speeds up the reaction rate 1. i.e. the time it takes to reach equilibrium ii. may change transiently (for a brief amount of time) 1. but catalysts remain unchanged in the overall process since it recycles to participate in another reaction 2. critical to understand in enzymatic reactions -- transient changes through interaction with substrate a. at the end, it emerges the exact same as it was before the reaction i. they are put back together (having an identical active site to react again) 1. recycle to interact in another reaction iii. differ from ordinary chemical catalysts in several important ways 1. higher reaction rates a. rates of enzymatically catalyzed reactions are typically 10 to 20 10 greater than those of the corresponding un-catalyzed reactions b. several orders of magnitude greater than those of the corresponding chemically catalyzed reactions 2. milder reaction conditions a. temperatures below 100 degrees b. at atmospheric pressure c. at nearly neutral pH 3. greater reaction specificity a. greater degree of specificity with respect to the identities of both their substrates (reactants) and their products than do chemical catalysts i. lock and key model ii. constitutive enzymatic reactions in cell 1. no point in enzymes rapidly producing products you dont need b. e.g. can exhibit stereospecificity i. exhibit stereospecificity 1. i.e. works on only one stereoisomer a. L amino acids  L specific enzymes c. can exhibit geometric specificity i. selective about the identities of the chemical groups of their substrate 1. important since end products are highly pure and are free of side (waste) products a. i.e. elimination of unnecessary waste products otherwise damaging to living cell i. vs chemical catalysts which may produce waste products ii. what is geometric specificity? 1. shapes are specific to whether or not substrate and enzymes can fit 4. capacity for regulation a. concentration of substances other than substrates and products can regulate activity i. allosteric control ii. covalent modification of enzymes iii. variation of amount of enzymes synthesized 1. up and down regulation b. enzymatic reactions need to sense (feedback control) when you need product and when you dont b. reactions that happen without presence of enzymes still occur i. but may take thousands of years 4. many enzymes do more than increase the rate of a single reaction a. they are often coupled to another reaction that can normally occur separately i. enzymes are coupled close together to increase efficiency ii. couple reactions 1. e.g. hydrolysis of ATP to ADP is often coupled to enzymatic reactions b. disc
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