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 general acid base is o Holoenzyme...

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 general acid base is o Holoenzyme (active) = Apoenzyme (inactive) + cofactor transfer of o Types: loosely bound to the enzyme cam be protons  Metal ions (Ca2+, Zn2+; inorganic ions) cycled on and off as the reaction between  Coenzymes (organic molecules) happens NAD+ is an example enzyme and sub Cosubstrates: loosely bound, cycle on & off specific Prosthetic groups: tightly/covalently bound tightly or covalently bound to the enzyme involves and remains attached an example is FAD water with Catalytic mechanisms acid: donation of a proton and stabilizes negative charge in transition(hydrolosis proton transfer 1. Acid-base catalysis esetrs & amides) o Partial proton donation (general acid catalysis) or abstraction (general base base: acceptance of proton and stabilizes the positive charge in metal ions catalysis). transition(deprotonation of alch and amines) stabilize neg 2. Covalent catalysis Enzyme active site has nucleophilic group charges on intermediates o Via transient formation of covalent E-S intermediate.that reacts with sub to form covalent they increase 3. Metal ion catalysis (and electrostatic catalysis) intermediate covalent inter is more stable than uncat electrophilicity o Enzymes that require metal ions for catalysis reaction of sub and o Metalloenzymes: tightly bound, usually transition metals electrostatic make them interactions more reactive o Metal-activated enzymes: loosely bound metal from solution, alkaline between they can 4. Proximity and orientation effect charged facilitate the o Binding to E forces substrate orient in a certain way  Rotational, translational groups in formation of motions cease  active groups face each other  reaction! enzyme and reactive species to participate in 5. Preferential binding of transition state (ES complex) su b can reaction o Affinity for E binding ES >> E+S or E+P (geometry) stabilize o Time at transition state ↑  [ES] ↑  chance to form product ↑ tran state es > e+s or e+p and lower Reaction order is given by its exponent bc geometry or activation interaction that and can o Order corresponds to molecularity of reaction (# of molecules collide). stabilize the help orient o For A+B  P+Q, V=k[A][B] transition state the sub  1st order for A or B alone. first order means the rate of reaction is linearly dependent on their  Overall, 2nd order concentrations 2nd means rate depends on product of the concentration of both more Stabil trans state the reactants more time spent leading to more es and more es is a 1 higher likelihood of forming product  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 weirdly backwards  Higher KM  weaker affinity o Vmax (maximum rate of the reaction) higher  Relates to Kcat and [Et] by Vmax = kcat[Et] max rate the max rate at which an enzyme can cat a reaction  Higher Vmax  higher max rate when the enzyme is saturated with sub means enzyme o kcat (turnover number or catalytic constant) can  Speed of catalysis process  Measures product formation (substrate turnover) turnover= number of sub to product per more sub  Higher kcat  higher speed enzyme molecule per unit time when un a enzyme is fully sat with sub given o kcat/KM is a measure of catalytic efficiency of enzyme. time when fully Michaelis-Menten equation when [s] is lower than km the reaction rate is approximately proportional to [s] sat o V = Vmax[S]/(KM+[S]) 1st order o If [S] = KM, when [s] is higher than km the rate approaches v mas 0 order  V = VmaxKM/(KM+ KM)  V = ½∙Vmax  The rate is half-maximal when [S] = KM. o If [S] = 2∙KM, v= reaction rate(velocity)  V = Vmax∙2KM/(KM+ 2KM) v max= max reaction rate  V = ⅔∙Vmax km= michaelis constant the sun o If [S] = 3∙KM, concentration at which the reaction rate is half of v max  V = Vmax∙3KM/(KM+ 3KM) [s]= sub con  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. increase in apparent km means higher con of sub is needed to reach half of v max 3 main types of reversible inhibition v max remains the same bc the inhibition can be overcome by increasing sub con 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). the presence of the inhibitor leads to a decrease in both the apparent km and v max 2  Copyright© by ChangHwan Lee Reproduc on or redistribu on of this material is prohibited without the author’s consent. o Noncompetitive (mixed) inhibition v max decreases due to the  Binds both E and ES (somewhere, not in the active site). fact the inhibitor effects to  Noncompetitive inhibitor changes the slope steeper. enzyme regardless of the  When KI = K’I sub con 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: prevents over production of the final  Inhibited by the final product (D) product which helps to maintain  Homeostasis: balancing [D] with the cell's needs. homeostasis  e.g., Thr dehydrogenase (synthesis of Ile). o Product inhibition: the product of an enzyme catalyzed reaction inhibits the enzyme that produced it  Inhibited by an immediate product this happens when the product binds to the  e.g., hexokinase by glucose-6-P enzymes active site preventing further sub binding and reaction it prevents over production of a Allostery product o Modulator binding to enzyme  different shape allostery effector molecule binds a o Cooperativity for complex diff site then active  Conformational change in one subunit  Change in other subunits in site causing a complex  other subunits’ affinity to S ↑ conformational  e.g., hemoglobin (T→R transition) change that affects activity  Sigmoidal curve for V/[S] allosteric site is  Most allosteric effectors affect only KM, but some change Vmax. where it binds causing conform oxidation is loss of Oxidation-reduction (redox) reaction changes and increase protein electrons o Involves electron transfer between 2 molecules. activity or decrease increasing o One is oxidized (loses electrons). The other is reduced (gains electrons). it oxidation o Biological oxidation often Loses 2H+ + 2e- or gains O. state reduction 3 is gain of electrons 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 hexokinase cat  2 GAP → 2 pyruvate (CH3-CO-COO-) phosphorylation of  ATPs are synthesized in steps 7&10 by phosphoglycerate kinase (PGK) and glucose to glucose-6- pyruvate kinase (PK) via substrate-level phosphorylation. phosphate using ATP o 3 control points: irreversible (essentially unidirectional) reactions as phosphate donner (step 1) this conversion  Hexokinase (step 1) inhibited by glucose-6-phosphate Inhibited by its product catalyzes phosphorylation of fructose-6- helps cells utilize phosphate to fructose-1,6-bisphosphate using glucose for energy  Phosphofructokinase (step 3) atp as phosphate donor allosterically production Inhibited by ATP and citrate regulated ATP and citrate inhibit which slows Activated by AMP down glycolysis and activated by AMP ADP  Pyruvate kinase (step 10) and fructose-2,6-bisphosphate increase PFK Inhibited by ATP and acetyl-CoA activity promoting glycolysis when energy is low Activated by AMP and fructose-1,6-bisphosphate. pyruvate kinase catalyzes the final step of glycolysis converting phosphoenolpyruvate (pep) to pyruvate with the production of ATP this reaction is essential for the generation of ATP it has allosteric reg fructose-1, 6-bisphosphate which activates ATP and alanine decrease activity when 4energy levels sufficient 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