Introduction to Enzyme Kinetics

Enzyme Kinetics Introduction to Enzyme Kinetics: Take reaction: A → B | Rate = k[A] (the rate is simply the rate constant k multiplied by the reactants) In order to increase the rate, you want to increase the concentration of the reactants The Vmax is simply the maximum rate at which the enzyme can catalyze reactions (assuming the substrate has saturated all enzymes present) if [E] remains constant If you want to increase Vmax then you need to increase [E] We must assume that throughout this, the natural conversion S → P is negligible when compared to the enzyme catalyzed reaction Steady State & the Michaelis Menten Equation: Steady State Assumption: the [ES] (intermediate) is constant and that any formation of ES is due to the loss of ES (AKA formation of ES = loss of ES) Remember, if Vo = Vmax then ALL enzyme is bound and is therefore in an ES state Michaelis Menten equation → If km = [S], then Vo = Vmax / 2 km is defined as the Michaelis Menten constant and is defined as the [S] where Vo = Vmax /2 Therefore the lower the km, the better the enzyme is at working when [S] is low kcat is also known as the enzyme’s turnover number (how many substrates an enzyme can turn to product per one second → units of sec-1) A higher kcat or a lower km while lead to an increase in the enzyme’s catalytic efficiency (a score of how good an enzyme is at speeding up reactions) Cooperativity: Cooperativity: is when substrate binding changes the substrate’s affinity (sigmoidal curve) This is backwards from what we’d normally think because we assume as less binding sites are available it is harder for substrate to bind Positive Cooperative Binding: substrate binding INCREASES affinity for subsequent substrate binding - hill coefficient greater than 1 Negatively Cooperative Binding: substrate binding DECREASES affinity for subsequent substrate binding - hill coefficient less than 1 Non-cooperative Binding: substrate binding has NO effect on affinity for subsequent substrate binding - hill coefficient equal to 1 Remember: cooperative binding can only be seen after substrate has binded to the enzyme and therefore is why in the graph to the right, at low [S] there isn’t much difference in binding affinity Hemoglobin (Hb) exhibits positive cooperative binding (4 binding sites) while Myoglobin (Mb) exhibits non-cooperative binding since it only has one binding site Also, PO2 is used instead of [O2] because oxygen is a gas (making pressure a more accurate depiction of substrate concentration) Allosteric Regulation & Feedback Loops: Allosteric Site: basically any spot other than the active site where enzyme regulators can bind to (there can be any number of them as well) Allosteric inhibitors or activators affect enzymatic activity by influencing the Vmax value or the km value Feedback Loop: when you have downstream products regulating upstream reactants Homotropic Regulator: when the substrate for the enzyme and the regulator for the enzyme are the same molecule (such as ATP for phosphofructokinase) Heterotropic Regulator: when the substrate for the enzyme and the regulator for the enzyme are different molecules (such as AMP for phosphofructokinase) NOTE: the best control steps (or committing steps) in enzymatic reactions are those which have very negative 𝛥𝐺° values (such as the phosphofructokinase step which has a 𝛥𝐺° value of around -4.5 kcal/mol) Non-enzymatic Protein Function: Main function of proteins are to bind specifically and tightly to molecules If you see the suffix -ase it is likely an enzymatic protein Also, not all enzymes are either an enzyme or not, sometimes they are a mix of the two 4 Main Non-enzymatic Protein Types: 1. Receptors/Ion Channels: receptors bind ligands in order to generate some sort of chemical cascade within the cell and ion channels act as a pore through the cell allowing certain ions to pass through the lipid bilayer of the cell 2. Transport Proteins: responsible for binding small molecules and transporting them to other locations (need a high affinity for ligand when [L] is high) 3. Motor Proteins: very important for cellular motility (i.e. myosin) 4. Antibodies: protein components of the adaptive immune system whose main function is to bind foreign and target them for destruction (antigen in this case is the antibody’s ligand) Covalent Modifications to Enzymes: 3 Types of “small” post-translational modifications: 1. Methylation: involves the addition of a methyl group (-CH3) to a protein 2. Acetylation: involves the addition of an acetyl group to a protein (-COCH3) 3. Glycosylation: involves the addition of a sugar molecule to a protein Zymogen: inactive form of an enzyme that requires covalent modification in order to become active (digestive enzymes that the pancreas releases - i.e. trypsinogen) Allows prevention of the protein to break down proteins that it shouldn’t while being transported to its desired location “Suicde” Inhibition: covalently bind the enzyme and prevent it from catalyzing any other reaction and RARELY unbind the enzyme (AKA irreversible inhibition) Additional Notes: km= (slope)*(1/y-intercept)=(slope)(Vmax)