1. 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

2. Enzyme Action E + S [ES] E + P enzymes catalyze reactions by lowering the energy of activation (Ea)

3. 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 non- reactive atoms -allows an altered local environment: changes ionic strength, pH, adds or removes H-bonds to substrate

4. Historical Background 2100 BCCodex of Hammurabi-description of wine making 700 BCHomer’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 1700sRéaumur - studies on the digestion of buzzards- digestion is a chemical rather than a physical process Late 1800s 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 1903Henri – first successful mathematical model 1913Michaelis and Menten – NZ rate equation.... 1950s-1960sKoshland – “Induced fit” model 1965Monod, Wyman and Changeux – allosteric regulation

5. 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)

6. 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

7. 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

8. Properties of enzymes as catalysts-1 Catalytic power They may increase reaction rate by as much as 10 15 -fold 2H 2 O 2 2H 2 O + O 2 Rate (L/mol/s) No catalyst1 x 10 -7 Fe 2+ catalyst56 Catalase4 x 10 7 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

9. 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

10. 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 25 o C –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

11. Classification of Enzymes Enzyme Commission (EC, 1955) - IUBMB International Union of Biochemistry & Molecular Biology 4 digit Numbering System [1.2.3.4] 1 st one of the 6 major classes of enzyme activity 2 nd the subclass (type of substrate or bond cleaved) 3 rd the sub-subclass (group acted upon, cofactor required, etc...) 4 th a serial number… (order in which enzyme was added to list)

12. 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] CH 3 CH 2 OH + NAD + ---> CH 3 CHO + 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 + H 2 O XOH + HA) Alkaline phosphatase [EC 3.1.3.1] R-PO 4 + H 2 O ---> R-OH + H-PO 4

13. 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 + CO 2 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 + P i ) Isoleucine-tRNA ligase [EC 6.1.1.5] L-isoleucine + tRNA Ile + ATP ---> L-isoleucyl- tRNA Ile + ADP + PPi

14. 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

15. 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...

16. 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)