Biological Membrane Structure and Enzymes PDF

Royal College of Surgeons in Ireland Medical University of Bahrain Biological Membrane Structure and Enzymes Salim Fredericks  Biological Membrane Structure and Enzymes Learning Objectives 1. Describe the key structural components of the biological membrane 2. Explain the principal f...

Royal College of Surgeons in Ireland Medical University of Bahrain Biological Membrane Structure and Enzymes Salim Fredericks  Biological Membrane Structure and Enzymes Learning Objectives 1. Describe the key structural components of the biological membrane 2. Explain the principal functions of the ‘lipid bilayer’ of the biological membrane 3. Describe the interaction between proteins and lipids of the biological membrane 4. Describe the different modes of transport across the biological membrane 5. Discuss the classification of enzymes 6. Describe the effect of temperature and pH on enzyme activity 7. Explain basic enzyme kinetics (including inhibition) and enzyme regulation Membranes  Membranes and the cell Nucleus Smooth Endoplasmic Reticulum Rough Endoplasmic Reticulum Mitochondrion Lysosome Golgi apparatus Transport vesicle Plasma Membrane Compartmentalization Membranes form a permeability barrier, control the movement of molecules, confine particular molecules to particular places Separates activites of the cell into different areas Separates activities of the cell into different times Membrane bound organelles Nucleus Mitochondrion Endoplasmic Reticulum Golgi Apparatus Lysosome Peroxisome Functions of the Cell Membrane To define the boundary of a cell; demarcate extracellular from intracellular. To regulate what enters and leaves the cell. To mediate direct interactions with other cells 1. tissue/organ formation 2. cell-cell recognition (immunity) 3. migration To receive and respond to extracellular signals (nutrients, hormones, growth factors, neurotransmitters) Components of the cell membrane The fluid mosaic arrangement of lipids and proteins in the plasma membrane Principles of anatomy and physiology 12th Edn G. Tortora The Phospholipid Bilayer PP Phospholipids spontaneously form bilayers in aqueous solution: - Micelles - Vesicles - Self-sealing The Phospholipid Bilayer PP The hydrophobic nature of the lipid bilayer is essential to its function as a barrier. Because it is hydrophobic, hydrophilic molecules cannot pass through spontaneously. A, A micelle is a small, spherical C, A liposome is the prototype structure with a hydrophilic of a membrane-bounded surface and a hydrophobic core. vesicle. B, A bilayer is the prototype of It forms spontaneously from a a biological membrane. As in lipid bilayer. the micelle, the hydrophilic D, A monolayer forms at the head groups are on the surface interface between water and and the hydrophobic tails are air. buried in the center. E, A soap bubble consists of two monolayers enclosing a thin water film. Principles of Medical Biochemistry, (2012) Membrane lipids - phospholipids G Fatty acid L Y C E R Fatty acid Phosphorylated O alcohol L Polar (hydrophilic) region Nonpolar (hydrophobic) region Phospholipids phosphatidylethanolamine (PE) phosphatidylcholine (PC) phosphatidylserine (PS) phosphatidylinositol (PI) Sphingolipid sphingomyelin (SM) Lipid distribution is asymetric phosphatidylcholine (PC) phosphatidylethanolamine (PE) phosphatidylinositol (PI) sphingomyelin (SM) phosphatidylserine (PS) Membrane Lipids : Cholesterol Cholesterol is the most hydrophobic component of the membrane It modifies the fluidity of the membrane:  Fluid Mosaic model of the membrane Singer and Nicolson Science 175 720-731 1972 The membrane is fluid and molecules may diffuse laterally through it. (vertical movement is more difficult) The membrane is composed of lipids and proteins The membrane is not uniform: different areas may have different properties, forming a mosaic Figure: Singer and Nicolson Science 175 720-731 1972 Membranes are fluid because the lipids and many of the proteins are free to rotate and move sideways in their own half of the bilayer. Integral membrane proteins - proteins that span the membrane 1. Single alpha helix 2. Multiple alpha helices 3. Rolled up beta sheets Structure of transmembrane protein Aquaporin Hydrophobic residues shown in blue Hydrophilic residues shown in red Integral membrane proteins - functions Peripheral membrane proteins - proteins that are present on one side only Not as firmly embedded in the membrane as integral proteins. They associate more loosely with the polar heads of membrane lipids or with integral proteins at the inner or outer surface of the membrane. Peripheral membrane proteins – localization and function attachment to enzyme cytoskeleton - cytoplasmic face - extracellular face cell surface attachment to marker extracellular matrix Transport The inside of the call cannot be completely isolated from its surroundings Nutrients and other substances must enter e.g. sugars, amino acids, vitamins Waste products must leave e.g. CO2 The plasma membrane is a selectively isolated from surroundings permeable barrier : it allows some substances through, but other substances are prevented from passing through Nutrients enter, waste products leave, important molecules are kept inside (e.g. DNA, proteins) Transport across the membrane energy structural TRANSPORT requirements requirements none DIFFUSION none channel or carrier proteins ATP ACTIVE carrier protein ion gradient vesicles vesicle proteins BULK/ ATP membrane VESICULAR fusion Diffusion Passive extracellular Requires no energy oxygen space concentration gradient input – molecules equilibrium travel down concentration gradient Small hydrophobic cytoplasm molecules can simply pass through the membrane Hydrophilic molecules need the help of membrane proteins Active Transport Requires energy Moves molecules against concentration gradient Primary Secondary Group Translocation Bulk transport Primary active transport Example: Na+/K+ pump Moves sodium out of the cell Moves potassium into the cell Against concentration gradient Energy provided by hydrolysis of ATP  Secondary active transport Uses concentration gradient built up through primary Na+ concentration active transport Bulk Transport Receptor-mediated endocytosis Phagocytosis, pinocytosis, endocytosis, receptor mediated endocytosis, exocytosis Involves the movement of membrane-bound exocytosis vesicles that merge with the membrane Ennzymes Enzymes Almost all chemical reactions that occur in the body are catalyzed by enzymes Globular proteins with primary, secondary and tertiary structure The catalytic cycle E+S ES EP E+P Classification Enzymes are classified by the reactions they catalyze Recommended name Usually substrate-ase glucose-6-phosphatase removes phosphate from G6P hexokinase adds phosphate to hexoses Trypsin …. cleavage of peptide bonds after Arg or Lys Classification Systematic name IUBMB divides enzymes in 6 major classes (subclasses, sub-sub classes etc) Complete description of reaction catalysed–ase e.c. number describes class and subclasses Hexokinase: ATP:D-hexose 6-phosphotransferase e.c. 2.7.1.1 Classification of enzymes Enzyme kinetics Reaction rate Increase in product concentration (or amount) per unit time Decrease in substrate concentration (or amount) per unit time Factors affecting reaction rate Temperature – increase in temperature: – as temperature initially rises - rate of reaction increases – reaction rate doubles with every 10 °C – exceed optimum temperature, rate falls rapidly pH pH All enzymes pH sensitive pH optimum reflects normal environment - e.g. stomach - acid pH liver - neutral pH pH Change pH – alters ionisation state of R groups – affects enzyme substrate interaction – affects 3-D structure Extreme pH – denaturation and precipitation – loss of enzyme activity Substrate concentration Initial rate Reaction rate or velocity is the number of substrate molecules converted into product per unit time Reaction progress curve Rate increases with [S] until a maximum velocity is reached – Vmax Most enzymes have a hyperbolic kinetic curve: graph of V vs [S] Allosteric enzymes often have sigmoid kinetic curves Saturation kinetic curve Saturation Kinetics Michaelis-Menten kinetics Kinetics k1 k2 E+S ES E+P k-1 Km = Michaelis constant = substrate concentration at ½ Vmax = (k-1 + k2 )/k1 Km approximates to a measure of the affinity of the enzyme for the substrate high Km : low affinity low Km : high affinity Michaelis-Menten Equation vmax [S] Vo = Km + [S] Assumptions: [S] >> [E] [ES] does not change (steady-state) only initial velocity is considered (V0) Relevance? Km tells us about enzyme’s affinity for substrate V0 is proportional to [E] at all values of [S] [S] > Km, V = Vmax Zero-order kinetics These equations and kinetic characteristics allow us to : Characterize and compare enzymes Characterize and compare substrates Characterize and compare inhibitors Double reciprocal plot vmax [S] Vo = Km + [S] Lineweaver-Burke plot: 1 KM 1 = + Vo Vmax[S] Vmax y = mx + c Lineweaver-Burke plot Lehninger principles 2nd ,box 8.1 Enzyme inhibitors Many drugs act by inhibiting enzymes Irreversible inhibitors Covalent bonds with enzyme Reversible inhibitors Non-covalent competitive non-competitive (uncompetitive) Competitive inhibition Inhibitor binds to same site as substrate, competes for access Increasing [S] will reverse the effect ie at high enough [S], velocity will reach Vmax More substrate is needed to reach ½ Vmax ie apparent Km is increased Reversible Inhibition - Competitive inhibitor Lehninger principles 2nd, box 8.2 Non-competitive inhibition Inhibitor and substrate bind to different sites (inhibitor can bind to E or ES) Increasing [S] has no effect ie Vmax is reduced by inhibitor Km is unchanged – the inhibitor does not change the affinity of E for S Reversible Inhibition - Non-competitive inhibitor Lehninger principles 2nd, box 8.2 Inhibitors - examples Competitive: Eg. Atorvastatin (Lipitor) inhibits HMGCoA reductase, key enzyme in cholesterol biosynthesis Eg Ritonavir – anti HIV inhibits HIV protease – enzyme involved in processing viral proteins Non-competitive Eg. Lead – environmental toxin Inhibits ferrochelatase, enzyme involved in haeme biosynthesis Allosteric Enzymes Define: allostery “In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site (that is, a site other than the protein’s active site)” Homotropic effector: the substrate itself Heterotropic effector : something other than the substrate Allosteric enzymes Regulated by the binding of small molecules to sites other than the active site – allosteric effectors Often key enzymes in metabolic pathways Enzymes with quaternary structure ie more than one polypeptide chain (generally) When effector is homotropic: Kinetic curve ( plot of V vs [S]) is sigmoid - for enzymes with more than one active site ie binding of substrate to one active site affects the activity of the other active site(s) Does not follow Michaelis-Menten kinetics