Enzyme Kinetics and Inhibition

• PHAR 284 Lecture 2 covers Bioenergetics & Metabolism, focusing on enzyme kinetics, inhibitors, regulatory enzymes, the role of ATP, and the Krebs cycle.

Enzyme Kinetics

• During reaction product formation, a high-energy transition state intermediate (T*) forms.
• The free energy of activation is the energy difference between reactants and T*.
• Lowering the free energy of activation increases the reaction rate.
• Enzymes catalyze biochemical reactions by offering an alternate pathway with lower activation energy, without changing reactant or product energies.

Factors Affecting Reaction Velocity

• Initial velocity (Vo) of enzyme-catalyzed reactions increases with substrate concentration [S] until reaching maximal velocity (Vmax) at saturation.
• Michaelis constant (Km) is the substrate concentration at 1/2 Vmax.
• Km signifies an enzyme's affinity for its substrate; it does not change with enzyme concentration.
• Small Km indicates high affinity because less substrate is needed to reach 1/2 Vmax.
• A large Km indicates low affinity because more substrate is needed to reach 1/2 Vmax.
• The double-reciprocal or Lineweaver-Burk plot is a straight line obtained when plotting 1/Vo versus 1/[S].
• Reaction velocity increases with temperature until reaching peak velocity, beyond which denaturation decreases velocity.
• Optimal temperature for human enzymes is 35°C-40°C; denaturation starts above 40°C.
• pH affects reaction velocity as enzymes need specific ionized or unionized groups for catalytic activity.
• Extreme pH levels can lead to enzyme denaturation.

Enzyme Inhibitors

• Inhibitors reduce the velocity of enzyme-catalyzed reactions.
• Irreversible inhibitors bind via covalent bonds; reversible inhibitors bind via non-covalent bonds, classified as competitive or non-competitive.
• Competitive inhibition occurs when the inhibitor competes with the substrate for the same active site via reversible bonding.
• In competitive inhibition, Vmax is unaffected because high substrate concentrations can reverse the inhibitor's effect.
• Km apparently increases in competitive inhibition because more substrate is required to reach Vmax.
• Non-competitive inhibition occurs when the inhibitor and substrate bind to different sites on the enzyme.
• Inhibitor binding does not prevent substrate binding, but reduces enzyme efficiency.
• Non-competitive inhibition cannot be overcome by increasing substrate concentration.
• In non-competitive inhibition, Vmax is reduced, but Km remains the same.

Examples of Drug Enzyme Inhibitors:

• Captopril (antihypertensive) is a competitive inhibitor of angiotensin-converting enzyme.
• Methotrexate (anticancer) is a competitive inhibitor of dihydrofolate reductase.
• Atorvastatin (cholesterol-lowering) is a competitive inhibitor of HMG-CoA reductase.
• Efavirenz (antiviral) is a non-competitive inhibitor of reverse transcriptase.
• Aspirin (anti-inflammatory) is an irreversible inhibitor of cyclooxygenase (COX).

Regulatory Enzymes

• Regulatory or "key" enzymes are rate-limiting in metabolic pathways.
• The activity of regulatory enzymes is controlled by allosteric modulation, covalent modification, or alteration of enzyme synthesis.

Allosteric Modulation

• Allosteric modulators (small metabolites or cofactors) reversibly bind to allosteric sites.
• Allosteric binding induces conformational changes, increasing or decreasing Vmax or Km.
• Modulation can be positive (+) or negative (-).
• Allosteric modulators can be homotropic (the substrate itself is the modulator) or heterotropic (a different metabolite is the modulator).

Covalent Modification

• Enzyme activity is regulated by phosphorylation or dephosphorylation of OH groups carried out by protein kinase and phosphoprotein phosphatase respectively.
• Phosphorylation can increase or decrease the activity, specific to the enzyme.

Alteration of Enzyme Synthesis

• Cells regulate enzyme amount by altering synthesis rates (induction or repression), which affects existing enzyme molecules.
• Insulin promotes the synthesis of key enzymes in glucose metabolism when blood glucose increases; this is a slow process.

Isoenzymes

• Isoenzymes (isozymes) are proteins catalyzing the same reaction but have different structures due to different amino acid sequences.
• Isoenzymes possess different charges; they are seperated using electrophoresis.
• They form of different subunits in various combinations
• They differ in kinetic/regulatory properties

Isoenzyme Examples

• Glucokinase & hexokinase catalyze glucose phosphorylation in different organs.
• Creatine kinase exists as 3 isoenzymes (CK1, CK2, CK3) formed from combinations of B and M subunits.
• CK1 (BB) is abundant in the brain and smooth muscle.
• CK2 (MB) is abundant in cardiac muscle.
• CK3 (MM) is abundant in skeletal and cardiac muscle.
• The isoenzyme pattern in plasma helps identify tissue damage sites and creatine (CK) via CK2 commonly diagnoses myocardial infarction.

Enzymes of Clinical Diagnostic Value

• "Functional enzymes" always circulate to perform a physiologic function (e.g., blood clotting).
• "Non-functional enzymes," from normal cell turnover, have no known function in blood and elevated levels signify tissue damage.
• Examples, aminotransferases diagnose myocardial infarction/viral hepatitis.
• Amylase indicates acute pancreatitis
• Creatine kinase indicates muscle disorders and myocardial infarction
• Lactate dehydrogenase is an indicator of myocardial infarction
• Lipase indicates acute pancreatitis

Bioenergetics & the Role of ATP

• Bioenergetics examines energy changes in biochemical reactions; thermodynamics predicts if a reaction can occur.

Free Energy

• The direction of a chemical reaction depends on enthalpy (ΔH) and entropy (ΔS).
• Enthalpy (ΔH) measures the change in heat content.
• Entropy (ΔS) measures the change in randomness.
• The free energy change (ΔG) is determined using ΔG = ΔH – TΔS, where T is absolute temperature in Kelvin.
• If ΔG is negative, the reaction is spontaneous and exergonic (net energy loss).
• If ΔG is positive, the reaction requires energy input and is endergonic.
• If ΔG = 0, the system is in equilibrium.
• Individual reaction ΔGs are additive in metabolic pathways, coupling unfavorable reactions to highly exergonic reactions to achieve a negative overall ΔG.
• Standard free energy change (ΔG°) is the ΔG under standard conditions.

ATP as an Energy Carrier

• ATP (adenosine triphosphate) serves as the cell's energy currency, linking catabolism and anabolism.
• Energy from nutrient catabolism makes ATP from ADP and inorganic phosphate (Pi).
• ATP donates chemical energy to endergonic processes, including metabolic synthesis, transport against concentration gradients, and muscle contraction.
• ATP contains two high-energy bonds. Cleavage of the phosphate group transfers substantial energy to an acceptor molecule.
• Major ATP sources are glycolysis, the citric acid cycle (Krebs cycle), and the respiratory chain.

Cellular Respiration

• Cellular respiration, occurring in cells, involves consuming O2 and producing CO2.
• It occurs in three main stages: acetyl-CoA production, acetyl-CoA oxidation, and electron transfer/oxidative phosphorylation.
• Stage 1: Organic fuel molecules oxidize to yield acetyl-CoA (glucose makes pyruvate, then acetyl-CoA via oxidative decarboxylation).
• Stage 2: Acetyl-CoA enters the citric acid (Krebs) cycle, reducing co-enzymes NADH and FADH2.
• Stage 3: Reduced enzymes enter the respiratory chain, reducing O2 to H2O and producing ATP (oxidative phosphorylation).

Oxidative Decarboxylation of Pyruvate

• Oxidative decarboxylation of pyruvate converts the α-keto acid pyruvate(glycolysis end-product) to acetyl-CoA in the mitochondrial matrix.
• This irreversible process is catalyzed by the pyruvate dehydrogenase complex.
• The pyruvate dehydrogenase complex consists of 3 enzymes (E1, E2, E3) and 5 co-enzymes.
• E1 is pyruvate dehydrogenase.
• E2 is dihydrolipoyl transacetylase.
• E3 is dihydrolipoyl dehydrogenase.
• The 5 co-enzymes are TPP (thiamine pyrophosphate), lipoate, co-enzyme A (CoA-SH), FAD, and NAD+.

Regulation of the Pyruvate Dehydrogenase Complex

• The pyruvate dehydrogenase complex is a metabolic gateway between glycolysis and citric acid cycle turned "ON" or "OFF" based on the cell's metabolic state.
• It is inhibited by its products, acetyl-CoA and NADH (negative feedback).
• Enzyme complex phosphorylation decreases activity, while dephosphorylation increases activity (covalent modification).
• Phosphorylation occurs when [Acetyl-CoA] / [CoA], [NADH] / [NAD], or [ATP] / [ADP] ratios increase, indicating abundant energy.
• Arsenite and mercuric ions inhibit the enzyme complex leading to lactate accumulation causing fatal lactic acidosis.
• TPP deficiency inhibits the enzyme complex, resulting in fatal lactic acidosis.
• Oxidative decarboxylation also converts α-ketoglutarate to succinyl-CoA in the citric acid cycle via α-ketoglutarate dehydrogenase complex.

Citric Acid Cycle (Krebs Cycle)

• Citric acid cycle, also tricarboxylic acid cycle (TCA), or Krebs cycle.
• Common end-metabolite, acetyl-CoA, reacts with oxaloacetate to form citrate and releases reduced co-enzymes, releasing CO2 and helps regenerate of oxaloacetate.
• Reduced co-enzymes oxidize in the respiratory chain for ATP ( Oxidative Phosphorylation )
• Matrix of mitochondria is the location in which the krebs cycle mainly lives in

The Krebs cycle has 8 steps

• Formation of Citrate ( Condensation of Acetyl-CoA with Oxaloacetate, catalyzed by citrate synthase. )
• Isomerization of Citrate to Isocitrate, catalyzed by aconitase
• Oxidative Decarboxylation happens forming Α-Ketoglutarate, catalyzed by isocitrate dehydrogenase
• Oxidative Decarboxylation happens forming succinyl-CoA. catalyzed by using 5 enzymes with pyruvate dehydrogenase complex.
• High-Energy cleaves and ATP makes which gets catalyzed by succinyl-CoA synthetase. Succinate Thikonase. Reaction helps with substrate level phosphorylation.
• Oxidation of Fumarate, catalyzed by succinate dehydrogenase
• Hydration of malate catalyzed by fumarase.
• Oxidation of malate to citrate, catalyzed by malate dehydrogenase, final of NADH is released

Vitamins involved in Krebs Cycle

• Niacin
• Riboflavin
• Thiamine
• Pantothenic acid

Krebs Cycle Importance.

• Serving in both catabolic and anabolic pathways
• Produces ATP through oxidation
• Supports intermediate Compounds
• Disposal of Fumarate

Inhibitors of the Krebs Cycle

• Citrate Synthase
• Aconitase
• A Ketoglutarate
• Succinate Dehydrogenase

Regulation of enzymes of the Krebs Cycle

• Citrate Synthase
• Isocitrate Dehydrogenase
• A KetoGlutarate

Energy yield of the Krebs Cycle

• Energy Yield ( 12 ATP is produced per turn )

Explore enzyme kinetics, including factors affecting reaction rates like temperature and pH. Understand enzyme inhibition, distinguishing between reversible and irreversible inhibitors and their impact on Km and Vmax. Also, learn the benefits of Lineweaver-Burk plots.