Study Guide for ABIO 365 Final Exam PDF

Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. Study guide for ABIO 365 Final Exam Enzymes Cofactors o Holoenzyme (active) = Apoenzyme (inactive) + cofac...

Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. Study guide for ABIO 365 Final Exam Enzymes Cofactors o Holoenzyme (active) = Apoenzyme (inactive) + cofactor o Types:  Metal ions (Ca2+, Zn2+; inorganic ions)  Coenzymes (organic molecules) Cosubstrates: loosely bound, cycle on & off Prosthetic groups: tightly/covalently bound Catalytic mechanisms 1. Acid-base catalysis o Partial proton donation (general acid catalysis) or abstraction (general base catalysis). 2. Covalent catalysis o Via transient formation of covalent E-S intermediate. 3. Metal ion catalysis (and electrostatic catalysis) o Enzymes that require metal ions for catalysis o Metalloenzymes: tightly bound, usually transition metals o Metal-activated enzymes: loosely bound metal from solution, alkaline 4. Proximity and orientation effect o Binding to E forces substrate orient in a certain way  Rotational, translational motions cease  active groups face each other  reaction! 5. Preferential binding of transition state (ES complex) o Affinity for E binding ES >> E+S or E+P (geometry) o Time at transition state ↑  [ES] ↑  chance to form product ↑ Reaction order is given by its exponent o Order corresponds to molecularity of reaction (# of molecules collide). o For A+B  P+Q, V=k[A][B]  1st order for A or B alone.  Overall, 2nd order 1 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. 4 kinetics quantities o KM (Michealis constant)  Enzyme binding affinity  Higher KM  weaker affinity o Vmax (maximum rate of the reaction)  Relates to Kcat and [Et] by Vmax = kcat[Et]  Higher Vmax  higher max rate o kcat (turnover number or catalytic constant)  Speed of catalysis  Measures product formation (substrate turnover)  Higher kcat  higher speed o kcat/KM is a measure of catalytic efficiency of enzyme. Michaelis-Menten equation o V = Vmax[S]/(KM+[S]) o If [S] = KM,  V = VmaxKM/(KM+ KM)  V = ½∙Vmax  The rate is half-maximal when [S] = KM. o If [S] = 2∙KM,  V = Vmax∙2KM/(KM+ 2KM)  V = ⅔∙Vmax o If [S] = 3∙KM,  V = Vmax∙3KM/(KM+ 3KM)  V = ¾∙Vmax Lineweaver-Burk plot (double reciprocal plot) o Slope: KM/Vmax o X intercept: -1/KM o Y intercept: 1/Vmax o Can determine KM and Vmax graphically. 3 main types of reversible inhibition o Competitive inhibition  Structurally similar to S  competes with S for active site binding.  Apparent KM ↑ but Vmax stays the same. o Uncompetitive inhibition  Binds only ES (not to the active site), but not to free enzyme.  Both KM and Vmax change  Does not change the slope (KM/Vmax). 2 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. o Noncompetitive (mixed) inhibition  Binds both E and ES (somewhere, not in the active site).  Noncompetitive inhibitor changes the slope steeper.  When KI = K’I o Y-intercept moves up, but X-intercept stays the same. o Apparent Vmax ↓ but KM stays the same. Metabolism Regulations on metabolic pathway o Feedback inhibition:  Inhibited by the final product (D)  Homeostasis: balancing [D] with the cell's needs.  e.g., Thr dehydrogenase (synthesis of Ile). o Product inhibition:  Inhibited by an immediate product  e.g., hexokinase by glucose-6-P Allostery o Modulator binding to enzyme  different shape o Cooperativity for complex  Conformational change in one subunit  Change in other subunits in complex  other subunits’ affinity to S ↑  e.g., hemoglobin (T→R transition)  Sigmoidal curve for V/[S]  Most allosteric effectors affect only KM, but some change Vmax. Oxidation-reduction (redox) reaction o Involves electron transfer between 2 molecules. o One is oxidized (loses electrons). The other is reduced (gains electrons). o Biological oxidation often Loses 2H+ + 2e- or gains O. 3 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. o Biological reduction often Acquires 2H+ + 2e- or loses O. Oxidation states of carbon in biomolecules o CO2: the most oxidized form of carbon found in living organisms. o In C-H bonds, the more electronegative C “owns” the 2e- shared with H. o In C-O bonds, O owns 2e-. Glycolysis o Takes place in the cytosol. o Glucose + 2ADP + 2Pi +2NAD+ → 2 pyruvate + 2ATP + 2NADH + 4H+ + 2H2O o Stage 1: Investment  Spend 2 ATP for glucose activation  1 glucose → 2 glyceraldehyde-3-phosphate (GA-3-P or GAP; triose phosphate)  ATPs are consumed in steps 1 and 3 by hexokinase (HK) and phosphofructokinase (PFK). o Stage 2: Payoff  Yields 4 ATP  2 GAP → 2 pyruvate (CH3-CO-COO-)  ATPs are synthesized in steps 7&10 by phosphoglycerate kinase (PGK) and pyruvate kinase (PK) via substrate-level phosphorylation. o 3 control points: irreversible (essentially unidirectional) reactions  Hexokinase (step 1) Inhibited by its product  Phosphofructokinase (step 3) Inhibited by ATP and citrate Activated by AMP  Pyruvate kinase (step 10) Inhibited by ATP and acetyl-CoA Activated by AMP and fructose-1,6-bisphosphate. 4 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. 3 fates of pyruvate 1. Aerobic conditions Pyruvate: o Processed through the TCA cycle (or citric acid cycle or Krebs cycle) o Complete oxidation to CO2 o Production of additional NADH and FADH2 2. Under anaerobic conditions 2a. Lactic fermentation o Coupled to reoxidation of NADH → NAD+ (recycling) o In rapidly contracting skeletal muscles of animals o Lactic acid buildup  acidification  muscle cramps 2b. Alcoholic fermentation o Yeast makes ethanol. o Pyruvate is reduced. o Also recycles NADH from Step 6 of glycolysis (GAPDH reaction in glycolysis). 5 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. TCA cycle (Tricarboxylic acid cycle, Krebs cycle, or citric acid cycle) o Takes place in the mitochondrial matrix. o Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O ↔ 2CO2 + CoA-SH + 3NADH + FADH2 + GTP + 3H+ o Step 1  Allosterically inhibited Succinyl-CoA (product of step 4): Intracyclic regulator NADH (product from 3 different steps): Indicator of energetic status o Step 3 (1st oxidative reaction)  Allosterically regulated inhibited by ATP and NADH. activated by ADP and NAD+. Measures NAD+/NADH and ADP/ATP ratios (High ratio: "green light") o Step 4 (2nd oxidative reaction)  Allosteric regulation Activated by AMP Inhibited by NADH and succinyl-CoA o Step 6  Oxidation of succinate + reduction of FAD  Reduces FAD (not NAD+)  Succinate dehydrogenase (Only membrane-bound enzyme in TCA) is also a part of electron transport chain (ETC). 6 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. Substrate-level phosphorylation: o Phosphorylation of ADP into ATP at the expense of substrate (oxygen-independent) o The substrate reacts to form a product containing a high-energy bond. Oxidative phosphorylation: o Respiration-linked phosphorylation (consuming oxygen) o Takes place at the electron transport chain (ETC) in the mitochondria. o Electron transport provides energy to pump protons out of matrix. o Uses the electrochemical gradient formed across the inner mitochondrial membrane. o All ETC complexes (I, II, III, and IV) actively pump protons into the intermembrane space during electron transport Reduction potential (E ): tendency of a given reacting species to gain e- when paired with a standard. 7 Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. o Positive E : tendency to attract/accept e- (reduced) o Negative E : tendency to give up e- (oxidized) o Two half cells (redox couples):  e- always spontaneously flow from the half-reaction with more negative E o (donor) to more positive E o (acceptor).  ∆ E o = E o acceptor - E o donor (standard reduction potential difference) Electron Transport Chain (ETC) o NADH → Complex I → CoQ → Complex III → cytochrome c → Complex IV → oxygen o FADH2 → Complex II → CoQ → Complex III → cytochrome c → Complex IV → oxygen Mechanism for ATP synthase (changes in binding affinity) o 3 αβ protomers, 3 distinct sites, in 3 different conformations (states):  O state: open, inactive, cannot bind ADP  L state: loose binding, inactive  T state: tight binding, active o Step 1: ADP + Pi bind to L site. o Step 2: L → T; T → O; O → L o Step 3: ATP is release from O. o After 2 more steps, the enzyme returns to its original state. Phosphate/Oxygen (PO) ratio o Ratio of ATP synthesized / Oxygen reduced o  4 protons should move down the gradient to make 1 ATP. o Movement of 2e- (1 ½O2) through ETC results in pumping out 10H+. o  P/O ratio is 10H+/4H+= 2.5 ATP molecules made per 2e- (1 ½O2). o If start from complex II, only 6H+ pumped out per 2e- transported. o P/O ratio is 6H+/4H+ = 1.5 ATP molecules per 2e-. 8