* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download lec1-introduction
Protein–protein interaction wikipedia , lookup
Multi-state modeling of biomolecules wikipedia , lookup
Adenosine triphosphate wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Lipid signaling wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Metabolic network modelling wikipedia , lookup
Citric acid cycle wikipedia , lookup
Western blot wikipedia , lookup
Restriction enzyme wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Nicotinamide adenine dinucleotide wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Biochemistry wikipedia , lookup
Biosynthesis wikipedia , lookup
Proteolysis wikipedia , lookup
Catalytic triad wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Metalloprotein wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Enzymes • They control metabolism by regulating metabolic reaction rates: molecules that accelerate or catalyze chemical reactions in cells by breaking old covalent bonds & forming new covalent bonds • Except for Ribozymes, all enzymes are proteins • a biological catalyst… • have complex structure (sequence of aa’s) • act only upon a specific substrate (or substrate group) • do not change the energetics of the reaction Enzyme Action E + S <---> [ES] <---> E + P enzymes catalyze reactions by lowering the energy of activation (Ea) What does an ES Complex do? - holds substrate out of aqueous solution holds substrate in specific orientation, close to Transition State to allow reaction to occur reduces ability of free rotation & molecular collisions with nonreactive atoms allows an altered local environment: changes ionic strength, pH, adds or removes H-bonds to substrate Historical Background 2100 BC Codex of Hammurabi-description of wine making 700 BC Homer’s Iliad: As the juice of fig tree curdles milk, and thickens it in a moment though it be liquid, even so instantly did Paeeon cure fierce Mars 1700s Late 1800s 1903 1913 1950s-1960s 1965 Réaumur - studies on the digestion of buzzardsdigestion is a chemical rather than a physical process Kühne - term 'enzyme': Greek "in yeast" Hans & Eduard Buchner – filtrates of yeast extracts could catalyse fermentation! No need to living cells E. Fischer – “lock and key” hypothesis Henri – first successful mathematical model Michaelis and Menten – NZ rate equation.... Koshland – “Induced fit” model Monod, Wyman and Changeux – allosteric regulation Terminology Many enzymes require a non-protein component for activity: • cofactor: small inorganic ions... mostly metal ions: Cu (cytochrome oxidase), Mg (kinases), Fe (catalase, peroxidase) • coenzymes: small non-protein but organic compounds Coenzyme A: acyl transfer Flavins: redox reaction NAD+ (NADP+): redox reactions Vitamins: derivatives of B vitamins (B1, B2, B6, B12), niacin, folic acid, riboflavin • prosthetic group: tightly bound large complex organic molecules, (heme) Holoenzyme vs apoenzyme (apoprotein) • active site: portion of enzyme which folds to precisely fit the contours of a substrate via weak electrostatic interactions & facilitates bond reactivity • allosteric site: a site other than the active site Isoenzymes • Classification is based on reaction catalyzed so enzymes isolated from different organisms but catalysing same rxn have same number but different amino acid sequence • Even within a single species, there may exist different forms of enzyme catalysing the same reaction. Differences may be: – A.acid sequence – Some covalent modification – 3-D structure • Isoenzyme (isozyme): different variants of the same enzyme having identical functions Properties of enzymes as catalysts-1 Catalytic power • They may increase reaction rate by as much as 1015-fold 2H2O2 2H2O + O2 No catalyst Fe2+ catalyst Catalase Rate (L/mol/s) 1 x 10-7 56 4 x 107 Specificity • Most enzymes are highly specific to their substrate and reaction catalysed – Bond specificity: e.g peptidase, phosphatase – Group specificity: e.g hexokinase – Absolute or near-absolute specificity • Stereospecificity: – Dehydrogenases catalyst the transfer of hydrogen from the substrate to a particular side of nicotinamide ring in NAD+ or NADP+ – Phenylalanine hydroxylase uses L-Phe not D-Phe • Importance of specificity in DNA replication and protein synthesis proofreading Properties of enzymes as catalysts-2 Regulation • Allosteric regulation (+/- effectors) e.g. feedback inhibition • Covalent regulation (phosphorylation by ATP-dependent protein kinases) e.g. Glycogen phosphorylase • Activation of zymogens, which are inactive proenzymes e.g. trypsinogen • Amount of enzyme: – gene expression – enzyme degradation How to define enzyme activity? Physical properties of an enzyme most often is measured by relative rate that substrate ---> product • 1 unit ACTIVITY= International unit (IU) amount enzyme which converts 1 μmole substrate per min at 25oC – e.g. IU= 10 μmole/min • 1 unit SPECIFIC ACTIVITY # IU of enzymatic activity per mg of total protein present – e.g. 10 μmole/min/mg protein or 10 IU/mg protein Classification of Enzymes Enzyme Commission (EC, 1955) - IUBMB International Union of Biochemistry & Molecular Biology 4 digit Numbering System 1st 2nd 3rd 4th [1.2.3.4] one of the 6 major classes of enzyme activity the subclass (type of substrate or bond cleaved) the sub-subclass (group acted upon, cofactor required, etc...) a serial number… (order in which enzyme was added to list) Major Classes of Enzymes-1 1. Oxidoreductases [dehydrogenases, oxidases, peroxidases] oxidation-reduction reactions, often using coenzyme as NAD+/FAD Alcohol dehydrogenase [EC 1.1.1.1] CH3CH2OH + NAD+ ---> CH3CHO + NADH + H+ 2. Transferases [kinase, phosphorylase, transaminases] group transfer reactions (AX + B BX + A) Hexokinase [EC 2.7.1.2] D-glu + ATP ---> D-glu-6-P + ADP 3. Hydrolases [digestive enzymes; amylases, lactase, sucrase] hydrolytic reactions: (AX + H2O XOH + HA) Alkaline phosphatase [EC 3.1.3.1] R-PO4 + H2O ---> R-OH + H-PO4 Major Classes of Enzymes-2 4. Lyases [decarboxylases] elimination rxns in which a double bond is formed Pyruvate decarboxylase [EC 4.1.1.1] pyruvate ---> acetaldehyde + CO2 5. Isomerases [mutases, cis-trans isomerases, racemases] isomerization rxns Alanine racemase [EC 5.1.1.1] L-alanine ---> D-alanine 6. Ligases [a.acid RNA ligase] condensation of 2 substrates at the expense of energy (ATP) (X + Y + ATP XY + ADP + Pi) Isoleucine-tRNA ligase [EC 6.1.1.5] L-isoleucine + tRNAIle + ATP ---> L-isoleucyl- tRNAIle + ADP + PPi Multienzyme systems • Proteins that exhibit more than one catalytic activity • EC recommendation more than one catalytic activity system e.g. fatty acid synthase system • Multifunctional enzymes will have more than one EC number... • Multifunctional enzyme can made up of: – Several polypeptide chains with different catalytic activities may be associated with each other – A single polypeptide chain with multiple catalytic site – or even both Tools of enzymology-1 Spectroscopic techniques (structure and reactivity in solution) • Optical (circular dichroism, UV-visible, fluorescence) • Vibrational (infrared, Raman) Electrochemical methods (kinetic analysis) • Potentiometric techniques • Conductometry Enthalpimetry (microcalorimetry) • Very sensitive and free of interference Radiochemical methods • Far more sensitive than photometric ones but... Tools of enzymology-2 X-ray crystallography • First crystallized enzyme, urease (J. Sumner, in 1926) crystals are proteins and their dissolution led to enzymatic activity • Within 20 years: >130 enzymes crystals documented • 3-D structure of a protein, myoglobin, was deduced (Kendrew, 1957) Multidimentional nuclear magnetic resonance (NMR) and X-ray crystallography are now commonly used: – to explain the mechanistic details of enzyme catalysis – to design new ligands Molecular Biology • Clone and express enzymes in foreign hosts (overexpression purification and characterization of enzymes occuring naturally in minute quantity) • Manipulate the a.acid sequence (site-directed mutagenesis and deletional mutagenesis chemical groups in ligand binding)