Lecture 12 (10/11/17) Lecture 12 (10/11/17)

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1 Lecture 12 (10/11/17) Reading: Ch1; Ch5; , Problems: Ch1 (text); 16 Ch4 (text); 1, 2, 3, 4, 6, 7, 8 NEXT Reading: Ch6; , Problems: Ch4 (text); 2, 3 OUTLINE Protein Characterization A. Collagen 1.4-S s 2.Biosynthesis 3.Disorders Lecture 12 (10/11/17) ENZYMES: Binding & Catalysis A. General B. Catalytic cycle; turnover number = k cat 1. Binding a. Models b. How? c. How tight? Binding curves i. Hyperbolic saturation ii. Sigmoidal cooperativity in saturation 2. Catalysis 1

2 Pro Pro-OH Gly (Gly-Pro/Ala-X) 330 X=hydroxyl-Pro, Glx, Arg, Asx, hydroxyl-lys (Cd), Ser Stability Melting (viscosity or CD vs. temp) cooperativity = sigmoidal plot role of hydroxyl-proline- T m Melting of Collagen Species Body Temperature Collagen T m Calf Shark, barracuda Cod, deep sea redfish % T m =39 CD vs. temp Higher T m correlates with higher OH-Pro/Pro 2

3 Modified Residues in Collagen & Elastin 1.4-9% 0.5-3% Prolylhydroxylase ox red Lysylhydroxylase (uses a similar mechnism) Scurvy: Vitamin C Deficiency 3

4 Cross-linking of Collagen Solubility: insoluble due to cross-linking lysyl oxidase à allysine LTQ cofactor merodesmosine Cross-linking of Collagen NH3 NH3 Allysine aldol Modified from Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg: Sponk (talk)translated by Sponk (talk) - Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg, CC BY-SA 3.0, 4

5 Cross-linking of Collagen NH3 NH3 Allysine aldol Modified from Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg: Sponk (talk)translated by Sponk (talk) - Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg, CC BY-SA 3.0, Protein Structure Collagen Collagen Biosynthesis 1) Transcription & translation 2) Protein modification hydroxylation glycosylation 3) quaternary structure & disulfides 4) Secretion 5) Procollagen processing 6) Cross-linking 7) Deposition 93 5

6 Diseases Collagen Disorders One thousand mutations have been identified in twelve out of more than twenty types of collagen. These mutations can lead to various diseases at the tissue level Scurvy caused by lack of Vitamin C needed for hydroxylating enzyme that makes Pro-OH (Hyp) & Lys-OH (Hyl) Osteogenesis imperfecta caused by a mutation in type 1 collagen weak bones and irregular connective tissue Ehlers-Danlos Syndrome caused by a mutation in type 3 collagen (EDS, type 4) Ten different types of this disorder that lead to deformities in connective tissue. Osteoporosis Not inherited genetically, brought on with age reduced levels of collagen in the skin and bone Knobloch syndrome Caused by a mutation in the collagen XVIII gene protrusion of the brain tissue and degeneration of the retina 6

7 ENZYMES (The most important class of proteins on Earth) General Properties of *t ½ = / k un (1 st order) k un = 0.693/t ½ 1. Enhance reaction rates 2. Mild reaction conditions 3. Reaction specificity 4. Regulated activity Rate Enhancement by * 1

8 General Properties of *t ½ = / k un (1 st order) k un = 0.693/t ½ 1. Enhance reaction rates 2. Mild reaction conditions 3. Reaction specificity 4. Regulated activity Rate Enhancement by * Pioneers Buchner and his brother Hans (of the funnel fame) was first to separate biological catalysis (fermentation) from living cells by trying to make jam using yeast extracts as a preservative, which they called a zyme. Enzyme was derived from in das zyme. Sumner was the first to isolate and crystalize an enzyme, urease. It was all protein, therefore enzymes=protein Haldane was the first really theorize how enzymes worked. 2

9 You can write out the catalysis as a series of reactions: But, this is better understood as a CATALYTIC CYCLE E + S ES EP E + P Divide the problem into two parts: Binding and Catalysis Substrate (S) Binding Enzyme (E) Enzyme Substrate Complex (ES) Substrate is bound at the active site Chemistry Enzyme Product Complex (EP) Product (P) The rate that an individual enzyme can go around this cycle is called the turnover number The turnover number is like a rate constant for catalysis, or k cat BINDING: Formation of the ES-complex Most do not interact with their substrates like a lock and key. Rather, the enzyme changes shape upon substrate binding, a phenomenon called induced fit Conformational change Lock-and-key model Induced-fit model (proposed by Emil Fisher) (proposed by Dan Koshland) A hybrid model is called ligand selective. This says that many conformations are in equilibrium in solution and the substrate binds one best, pulling equilibrium

10 BINDING: First demonstration of Induced Fit Hexokinase: Glucose + ATP Glucose 6-phosphate + ADP Hexokinase Glc 6-P Hexokinase Hexokinase: Glucose + ATP Glucose 6-phosphate + ADP 4

11 bring substrates together to form an enzyme-substrate complex on a particular region of the enzyme called the active site. ES-complex The interaction of the enzyme and substrates at the active site promotes catalysis. The protein fold brings residues important for Binding & Catalysis to the Active Site. A schematic representation of the primary structure of lysozyme shows that the active site is composed of residues that come from different parts of the polypeptide chain. Enzyme-Substrate Complex Drives Selectivity Chymotrypsin with bound substrate (PDB ID 7GCH to just observe the interactions of substrate and enzyme) 5

12 BINDING: great example of complementarity Example: Tyr-aminotransferase Weak interactions (non-covalent bonds) Hydrogen Bonds Salt bridges Van der Waals Hydrophobic effect This kind of complementarity is not restricted to ES complex: interactions of receptor-ligand, protein-protein, protein-nucleic acid, etc. What is the stereo-isomer bound here? L-Tyr BINDING: are stereo-selective...even for molecules that are not chiral (so-called pro-chiral) Example: Lactate Dehydrogenase(LDH) :H :H re-face (clockwise) si-face (counter clockwise) HO HO H C C H CH 3 COO COO CH 3 Pro-S H 3 C HO H C HO H C CH 3 COO L-Lactate (S) (counter clockwise) Pro-R COO D-Lactate (R) (clockwise) The hydride (:H )(from NADH) can add to the sp 2 carbonyl carbon from the top (on the re-face) or the bottom (si-face). But, it can only do ONE. 6