Biochemistry 2: Enzyme Enzymes as Biological Catalysts Effects of Local Conditions on Enzymes Enzymes: Biological catalysts that are unchanged by the reactions Temp and pH: they catalyze & are reusable. Enzymes DO NOT alter the DG or DH, nor the final equilibrium position. They only change the rate of reaction by altering the mechanism. Salinity: Catalyze both the FORWARD & REVERSE reactions. Exergonic Rxns: Release energy; DG is negative. Endergonic Rxns: Require energy; DG is positive. Oxidoreductases: REDOX reactions that involve the transfer of e-. Transferases: Move a functional group from one molecule to another. Hydrolases: Catalyze cleavage with the addition of H2O. Lyases: Catalyze cleavage without the addition of H 2O and without the transfer of e-. The reverse reaction (synthesis) is often more important biologically. Isomerases: Catalyze the interconversion of isomers, including both constitutional isomers and stereoisomers. Ligases: Join two large biomolecules, often of the same type. Lipases: Catalyze the hydrolysis of fats. Dietary fats are broken down into fatty acids and glycerol or other alcohols. Kinases: ADD a phosphate group. A type of transferase. Phosphatases: REMOVE a phosphate group. A type of transferase. Phosphorylases: Introduces a phosphate group into an organic molecule, notably glucose. Enzyme Kinetics Saturation Kinetics: As ![S] Þ !rxn rate, until a max value is reached. Graphical Plots: Michaelis-Menten: Hyperbolic curve Lineweaver-Burk: Line Km The [S] at which an enzyme runs at half its Vmax. % ($% 𝐾" =$ &'$ ) %' Vmax: The maximum rate at which an enzyme can catalyze a reaction. This is when all enzyme active sites are saturated with substrate. Michaelis–Menten 𝑉 =𝑉 ",-$ + Equation: [/] [ / ] (%1 Cooperative Display a sigmoidal curve because of the change in Enzymes: activity with substrate binding. Mechanisms of Enzyme Activity Enzymes act by stabilizing the transition state, providing a favorable microenvironment, or bonding with the substrate molecules. Active Site: The site of catalysis. Can affect an enzyme’s activity in vivo; changes in temperature and pH can result in denaturing of the enzyme and loss of activity do to loss of 2°, 3°, or 4° structure. In vitro, salinity can impact the action of enzymes. Regulation of Enzymes * See appendix for detailed information on enzyme inhibition Feedback Inhibition: An enzyme is inhibited by high levels of a product from later in the same pathway. Reversible The ability to replace the inhibitor with a compound Inhibition: of greater affinity or to remove it using mild laboratory treatment. Competitive When the inhibitor is similar to the substrate and Inhibition: binds at the active site, blocking the substrate from binding. Can be overcome by adding more substrate. Vmax is unchanged, Km increases. Uncompetitive When the inhibitor binds only with the enzymeInhibition: substrate complex. Vmax and Km both decrease. Noncompetitive When the inhibitor binds with equal affinity to the Inhibition: enzyme and the enzyme-substrate complex. Vmax decreases, Km is unchanged. Mixed Inhibition: When the inhibitor binds with unequal affinity to the enzyme and the enzyme-complex. Vmax decreases, Km is increased or decreased depending on if the inhibitor has a higher affinity for the enzyme or enzyme-substrate complex. Irreversible Alters the enzyme in such a way that the active site is Inhibition: unavailable for a prolonged duration or permanently. Suicide Inhibitor: A substrate analogue that binds IRREVSERIBLY to the active site via a covalent bond. Allosteric Effecter: Binds at the allosteric site and induces a change in the conformation of the enzyme so the substrate can no longer bind to the active site. Displays cooperativity, so it does not obey Michaelis-Menten kinetics. Positive Effectors: Exert a positive effect, !activity. Negative Effectors: Exert a negative effect, ¯activity. Homotropic Effector: An allosteric regulator that IS ALSO the substrate. Ex: O2 is a homotropic allosteric regulator of hemoglobin. Heterotropic Effector: An allosteric regulator molecule that is DIFFERENT from the substrate. Phosphorylation: Covalent modification with phosphate. Catabolism: Phosphorylated = active Anabolism: Phosphorylated = inactive Glycosylation: Covalent modification with carbohydrate. Zymogens: Precursor to an enzyme. Secreted in an inactive form and are activated by cleavage. Lock & Key Theory:The enzyme and substrate are exactly complementary and fit together like a key into a lock. Induced Fit Theory: The enzyme and substrate undergo conformational changes to interact fully. Cofactors: Metal cation that is required by some enzymes. Coenzyme: Organic molecule that is required by some enzymes. Reaction scheme for Michaelis-Menten enzyme activity. To simplify things, we assume that almost none of the product reverts back to ES, which is true at the start of the reaction. This is why K-2 is omitted from the reaction scheme shown below.