Enzymes: Basic Principles SBS017 Basic Biochemistry Dr John Puddefoot Objectives: To introduce the basic concepts and definitions of enzymology You should be able to able to define the terms Enzyme, Specificity and Co-factor You will understand the concept of Gibbs Free Energy and its relation to reaction equilibrium You will be able to describe how enzymes effect the rate of biological reactions and be able to define the term Activation energy in the context of Transition state theory. Enzymes Biological catalysts Almost all enzymes are proteins (but RNA can have enzymic activity too, ribozymes) Function by stabilizing transition states in reactions Enzymes are highly specific Enzymes accelerate biological reactions e.g. Carbonic Anhydrase CO 2 + H 2 O H 2 CO 3 Each molecule of enzyme can hydrate 1,000,000 molecules of CO 2 per second, 10,000,000 times faster than uncatalysed reaction Catalase 2H 2 O 2 2H 2 O + O 2 Rate enhancement by enzymes Kcat - maximum number of enzymatic reactions catalyzed per second. Enzymes are highly specific Proteolytic enzymes Hydrolysis of esters Proteolytic Enzymes differ in degree of substrate specificity e.g Peptide bond hydrolysis (proteolysis) A. Trypsin cleaves only after arginine and lysine residues. B. Thrombin cleaves between arginine and glycine only in particular sequences. But Papain cleaves all peptide bonds irrespective of sequence Specificity is important e.g. DNA polymerase I Adds nucleotides in sequence determined by template strand. Error rate of < 1 in 1,000,000 Due to precise 3D interaction of enzyme with substrate Major classes of enzymes Many enzymes require cofactors Cofactors are small molecules essential for enzyme catalysis Can be: i.Coenzymes (small organic molecules) ii.Metal ions Holoenzymes Enzyme without its cofactor apoenzyme With its cofactor holoenzyme Cofactors are essential for activity e.g. many vitamins are cofactors, many diseases associated with vitamin deficiency due to lack of specific enzyme activity Enzyme cofactors Energy transformation Many enzymes transform energy into different forms Adenosine Triphosphate (ATP) is universal currency Light ATP Photosynthesis Food ATP Respiration ATP work ATP is an energy carrier e.g. ATP provides energy to pump Ca2+ across membranes Free energy The free energy of a reaction is the difference in free energy between its reactants and its products This called the G If G is negative, the reaction will occur spontaneously exergonic If G is positive, energy input is required endogonic: Endergonic reactions .. G is positive Exergonic reactions !G negative .. G is negative Exergonic reactions G is negative H2O2H2O2 2H 2 O 2 + O2 G is independent of reaction path A system is at equilibrium and no net change can occur if G is zero Calculating G The free energy of a reaction is given by: Where: G is the standard free energy change (i.e. change at 1M concentrations), R is the universal gas constant T the absolute temperature Calculating G at pH7 (G) At equilibrium and standard pH 7 And so Calculating G at pH7 ( G) Rearrange and substitute K eq or in log 10 Rearranges Example Calculate G and G At equilibrium GAP to DHAP is If DHAP is 2 x10 -4 M and GAP is 3 x M