Biochem exam III

• sole C source is CO2 in atmosphere
• energy source is light (plants, photosynthetic bacteria)

• C source is carbs, fats and proteins
• energy is degredation of biopolymers
• can be aerobes or anaerobes

require O2 for metabolism

don't require O2 for metabolism

• an entire series of steps, converting a precursor to product (like glycolysis converts glucose to pyruvate)
• Can be Linear, cyclic or spiral

• intermediates in the pathway (stable compounds) 
• most molecules inside living organism

• degrades complex molecules into small, simple molecules
• exergonic, releases energy (usually ATP)
• oxidative (uses NAD+ and FAD)

• synthesizes complex molecules from small molecules
• reductive (uses NADPH)
• requires energy, endergonic (ATP)

• catabolism (convergent): energy generating reactions (glucose oxidation) coupled to energy utilizing reactions (ATP synthesis)
• anabolism (divergent): energy generating reactions (ATP hydrolysis) coupled to energy utilizing reactions (biosynthesis, mechanical work)

• macromolecules
• monomers
• acetyl CoA

free energy change for a reaction.  the MAXIMUM AMOUNT of free energy that a reaction can deliver.  G of products - G of reactants

ΔG (free energy change for a reaction) at standard conditions (25 C, 1 atm, 1M solute concentration)

ΔG^o (free energy change for a reaction at 25C, 1atm, 1M solute) at pH 7, H2O 55.5M

-RT ln K'eq

• K'eq =  
• Can use equilibrium concentrations of rx components to calculate (0.200M, nothing for water)

negative, exergonic and spontaneous

positive, endergonic, nonspontaneous

0, nothing, at equilibrium

none

none

• ΔG at physiological conditions, different for each species.  
• 37C and physiologic solute concentration
• can be spontaneous even if ΔG^o' is not (spon in vivo)

• ΔGp = ΔGo' + RTlnQ
• Q = mass action expression, same as K'eq at NONEQUILIBRIUM

• Q (like K'eq but at NON-EQUILIBRIUM conditions)
• 

• the degree of disorder/randomness in a system
• gas has higher S than liquid
• mixture has higher S than two separate
• mix of amino acids has higher S than protein with same aas.  
• Constantly increasing (NaCl + H2O becomes Na + Cl, increase in S)

• ΔG = ΔH - TΔS
• free energy = enthalpy (heat content, - for exothermic) - temp (K) x entropy

• hydrolysis of high energy compound pays for anabolic rx with -ΔG^o'. 
• - must be larger than +

• contain one or more bonds whose ΔGo' of hydrolysis is more negative than -25 kJ/mol.  
• Hydrolysis is IRREVERSIBLE
• high activation energy for hydrolysis
• require enzymes to be broken down

• anhydrides (resonance) (ATP, lowest)
• mixed anhydrides (resonance)
• enoyl phosphate (PEP, highest)(keto-enol)
• phosphocreatinine (resonance)
• thioesters (acetyl CoA) (resonance

balance and split equilibrium reaction, add half-reactions from table, add ΔG^o' (flip the backwards one)

more unstable (more negative ΔG^o') to less unstable (less negative ΔG^o')

• max # of ATP formed from a catabolic step calculated by ΔGo' for that step
• 
• always round down to nearest WHOLE NUMBER.

gets more out of glc

• loss of electrons.  Oxidized thing is the reducing agent.  
• gain of O
• Loss of H

• gain of electrons.  Reduced thing is the oxidizing agent  
• Loss of O
• gain of H

• nicotinamide adenine dinucleotide
• becomes NADPH when P added to 2'OH of ribose
• from vit B3 (niacin)
• can follow on assays at 340nm

• flavin adenine dinucleotide
• absorbs light at 450nm
• derived from B2 (riboflavin)
• FAD - AMP = FMN (flavin mononucleotide)

• is spontaneous
• the ΔG^o' is negative (=-nFΔE^o')

• reduction potential (electric potential), in volts.  
• Highest on table = highest E^o'
• To find oxidizing agents that would be reduced by something, look ABOVE ON TABLE

• ΔE^o' = -nFΔE^o'
• n = # electrons transferred or # molecules in the rx
• F is a constant, given

When potential difference (ΔEo')(V) between two electron carriers in the electron transport chain is large enough to cause a ΔGo' that is MORE NEGATIVE than -30.5kJ/mol, can be chemically coupled to make 1 mole of ATP

• anaerobic oxidation of glucose, 2 steps (investment and payoff)
• glc + 2 ADP + 2Pi + 2 NAD+ →
• 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2H₂O

• anaerobic glycolysis (lactic acid fermentation)
• ethanol fermentation (yeast)
• citric acid cycle

• 2 CH₃-C-COO- + 2 NADH + 2 H+ →
• 2 CH₃-C-COO- + 2 NAD+
• source of muscle ATP during sustained muscle exercise, anaerobic (fast sprint, 10s) 
• stored ATP, creatine phosphate, then glycogen (slow)
• helps survive brief hypoxia
• only ATP in some areas of body (retina, cornea, RBC) b/c no mitochondria

• 2 pyruvate (CH3-C-COO-) + 2 NADH + 4H+ → 2 CO2 + 2 ethanol + 2 NAD+
• reoxidizes NADH to NAD+

acid and conjugate base.  Pyrvate and lactate are COO- form, at pH 7.0.

• ATP phosphorylates sugar
• glc is destabilized
• no oxidation/energy release
• 5 steps.

ATP produced.

• investment step
• kinase transfers phosphoryl, works on any hexase
• gatekeeper step, traps glc in cell, commits to metabolism
• activation step, irreversible, regulatory enzyme

• investment step
• aldose/ketose isomerization (isomerase), changes carbonyl group position
• positive ΔG^o'

• investment step
• kinase catalyzes irreversible, catalyzed by regulatory enzyme

• investment step
• lyase
• 6C sugar split into 2 3-C sugars, REVERSE ALDOL CONDENSATION (joining of aldehydes or ketones = alcohols)
• fru 1,6-bis-P unique to glycolysis

• investment step
• prevents glycolysis becoming 2 pathways (inefficent), lets all subsequent steps be x2
• joining of fork in pathway (triangle)

oxidoreductase that removes H, used in step 6 of glycolysis

• first payoff step
• not negative ΔG, coupled with step #7.  
• dehydrogenase = oxidoreductase that removes H
• energy from oxidation of substrate drives phosphorylation, produces high-energy mixed anhydride + NADH

• pay-off step
• high-energy, gives some energy to previous step (coupled) 
• Enzyme is only non-regulatory kinase (reversible)
• SUBSTRATE-LEVEL PHOSPHORYLATION (1,3BPG is higher energy than ATP)

isomerase that moves a group between 2 positions on same molecule

• payoff step
• mutase, moves Phosphoglycerate to another spot on same molecule

• payoff step
• dehydration, catalyzed by a lyase, removes H2O to make double bond, makes a high-energy compound (highest)

• payoff step
• very exergonic so irreversible/regulatory
• 2nd substrate-level phosphorylation
• last step

• net gain of 2 ATP per glc oxidixed (2 invested, 4 yield)
• 2 NADH produced from NAD+
• requires Pi

• rate of glycolysis decreases when muscles switch from anaerobic to aerobic metabolism
• Don't need to work as hard, so don't. 
• Feedback inhibition, only make as much ATP as you need.

2 ATP/glc

32 ATP/glc

enzymes of glycolysis are connected, CHANNEL SUBSTRATE from one step to next, funnel in, faster, eliminates diffusion.

pyruvate = acetyl CoA = citric acid cycle (+ ADP, Pi, NADH, H) = CO2 + NAD+ + ATP + H2O

• lactate fermentation (anaerobic glycolysis of animal muscle): pyruvate + NADH + H+ ⇄ LDH ⇄ lactate + NAD+
• alcohol fermentation: pyruvate + H+ → pyruvate decarboxylase, TPP → acetaldehyde + NADH + H+ ⇄ alcohol dehydrogenase ⇄ ethanol + NAD+

• recycling of lactate into pyruvate in liver after you catch your breath.  
• otherwise lactate is dead-end product, lowers pH of cells and causes cramping.  Just regenerates NAD+ to keep glycolysis moving.  REDOX, H+ eventually inhibits PFK-1 to avoid lactic acidosis

• shortage of substrate.  Aerobic turns NADH to NAD+, no NADH to run lactate synthesis.  
• Pyruvate sent into mitochondria so also not available.

• anaerobic pathway of pyruvate metabolism.  
• TPP as cofactor, nucleophile cleaves bonds acetyl group carrier, decarboxylates.  
• irreversible, releases CO2

• oxidized in liver into acetyl CoA, becomes fat or citric acid cycle.  
• excessive depletes NAD+ = liver cirrhosis
• accum of acetaldehyde (when no NAD+) causes hangover.  
• Inhibits glycolysis and gluconeogenesis, causes hypogylcemia and temp regulation issues

• duslfiram: accumulates acetaldehyde to increase hangover
• opioid antagonist: counteracts high, suicide.

acetaldehyde crosses placenta, fetal liver can't oxidize

• oxidized by ADH to formaldehyde
• tx with IV ethanol bc ADH is nonspecific ALCOHOL dehydrogenase, so competitive inhibition with higher-affinity substrate

• cellular glycogen glycogen phosphorylase PHOSPHOROLYTIC CLEAVAGE
• only works on 1->4 linkage, enzyme phosphoglucomutase for nonreducing end when hits 1->6
• isomerization sends into glycolysis
• glycogen cleavage MOBILIZES stored fuel (regulatory)

• hydolyzed by salivary and pancreatic a-amylases at a1->4
• isomaltase for a1->6
• monosacc enter glycolysis

• cleaved to monosacc by membrane-bound enzymes at brush border (isomaltase, maltase, sucrase, lactase), become glc, fru, gal
• (____ + H2+ ---> 2____)
• hexokinase works on any hexose.  Liver uses fructokinase
• galactokinase on UDP

• genetic disease, lacks enzyme in galactose pathway
• hepatomegaly, jaundice, cataracts, mental retardation, vomiting

• enzyme in feeder pathway for glycolysis
• degrades cellular glycogen (removes 1 glc and phosphorylates)
• works on a1->4

• enzyme in glycolysis and feeder pathways
• phosphorylates any hexose.  Fructose, mannose in feeder pathway, glc in glycolysis

energy charge = 

Maintain constant blood glucose, ATP, variation of priorities in dif cells, avoid futile cycles, keep metabolic intermediates from accumulating.  Blood glc wins for brain.

• allosteric regulation (feedback inhibition, feed-forward activation)
• covalent modification
• differential behavior at isozymic forms (myosites vs hepatocytes)

• distinct enzymes that catalyze the same reaction
• usu oligomeric enzymes, dif in subunit to make dif activities, Vmax, Km, pH optimum.
• LDH has H and M subunits, work in muscle and heart, etc.  Requires fewer genes to make

• isozymes have different Vmax, Km, pH optimum, etc.  Behaves differently based on location.  
• Other option is temporal regulation, different at different times.

• diagnose tissue damage
• usu intracellular, so if in blood, tissue damage
• electrophoresis vs ELISA

• KINASE, AMP = ON ------ PHOS A PHOS, ATP, GLC = OFF
• allosteric or covalent
• makes/uses ATP in muscle, breaks down glycogen to maintain blood sugar in liver

muscle activated by AMP, inhibited by ATP.  Liver inactivated by glc

• activated by cascade (adenylyl cyclase → ATP-cAMP activates PKA, activates phosphorylase b (conformational), glc becomes glc 1-P.  
• Signal amplification
• Off by phosphorylase a phosphatase and phosphodiesterase
• diff purposes in dif tissues

• mycyte: produce ATP in response to epinephrine, break down glycogen
• hepatocyte: maintain blood glc levels by breakdown of glycogen

• allosteric
• feedback inhibition by glc 6-P (intermediates)
• If PFK-1 decreases glc 6-P will build up too high, but need glc 6-P for glycogen so ISOZYMES.  Only active if high blood glc.  Weak inhibition, just higher K_0.5

• major regulatory enzyme in glycolysis after glycogen synthesis branch.  
• fru 1,6 bis-P UNIQUE to glycolysis
• allosteric, inhibited by products of glycolysis (ATP, citrate, H+)
• activated by AMP, ADP, fru-2,6-bis-P (increases affinity in liver to slow glycolysis when blood glc is low)

• allosteric: inhibited by ATP, acetyl CoA and alanine (like pyruvate, feedback inhibition)
• activated by fru 1,6-bisP (synch with PFK-1)
• covalent: liver isozyme inactivated with low blood glc
• Break even point in glycolysis
• most dramatic effect on flux through glycolysis (rest are stronger regulators)

• glycogen becomes glc 1-P, AMP allosteric activator of phosphorylase b
• glycolysis makes ATP, AMP allosteric activator of PFK-1

• all decrease.  In muscle glc 6-P increases, inhibits hexokinase
• in liver glc 6-P increases but does not inhibit glucokinase, glycogen is made

• glucagon decreases PFK-1 and pyruvate kinase
• glc 6-P dephosphorylates to increase glc in blood
• glucokinase decreases (high Km)
• glcyogen phosphorylase increases, glycogen broken down

• alternate use of glc, provides ribose 5-P, NADPH, uses ATP
• NADPH reduces tripeptide glutathione, which protects erythrocytes from oxidative damage
• glucose 6-P dehydrogenase deficiency: genetic X-linked dz, protects against malaria

• multienzyme complex of mitochondrial matrix
• receives pyruvate from cytosol, converts to acetyl CoA, uses NAD+ (reduced) and pyruvate (oxidized by oxidative decarboxylation)
• gatekeeper (aerobic oxidation), activation, regulatory
• 5 steps, 3 enzymes, 5 coenzymes

• 1. decarboxylation of pyruvate (TPP - acyl carry)
• 2. oxidation of activated aldehyde to thioester (lipoyllysine - acyl carry and oxidize)
• 3. transfer of acetyl group from lipo to CoA (acyl carrier and activator--trans, GOAL ACCOMPLISHED)
• 4. oxidation of lipoyllysine by FAD (recycle cofactors)
• 5. oxidation of FADH2 by NAD+ (recycle cofactors)

• CoA and NAD+ used as substrates (one in, one out)
• TPP, lipoyllysine and FAD used and regenerated

multiple copies of each component packed into a specific geometry that allows substrate channelling

2.  2 pyruvate enter cycle from glycolysis, 2 acetyl CoA, 2 CO2 exit.

• aldol condensation and hydrolysis
• reaction driven by hydrolysis of acetyl CoA
• irreversible, regulatory

• OH is tertiary, moves to oxidizable position
• 2 steps vial dehydration-rehydration
• aconitase is a LYASE (not a mutase)

• oxidative decarboxylation
• regulatory/irreversible

• oxidative decarboxylation
• like PDH complex
• Irreversible, regulatory
• GOAL ACCOMPLISHED, have thioester CoA, now regerate oxaloacetate

• substrate-level phosphorylation
• hydrolysis of high-energy "pays" for ATP/GTP (which depends on enzyme)

• ONLY redox that produces C=C
• stereospecific - TRANS only
• ONLY FAD in cycle
• ONLY membrane-bound enzyme in cycle
• part of electron transport chain, feeds FADH into it

• hydration, from lyase
• only L-isomer formed (stereospecific enzyme)

• redox with oxidoreductase
• regenerates starting compound

• 1 GTP, 3 NADH, 1 FADH2 per acetyl CoA
• 2 acetyl CoA/glc
• 10 ATP per acetyl CoA
• 20 ATP per glc 
• doesn't include PDH or glycolysis

PDH complex, feeds acetyl CoA into cycle

• high ratio  of ATP/ADP, NADH/NAD+, acetyl CoA/CoA
• FEEDBACK INHIBITION

• allosterism
• covalent by reversible phosphorylation

• allosteric
• inhibited by high energy charge (ATP, NADH)
• activated by low energy charge (ADP, NAD+) (Not FAD)
• coordinates with glycolysis, citrate inhibits PFK-1