CELLULAR ENERGETICS            Feb 12 - Darwin's BDay
            
read chapter 12/6e                                              Sep 25 - Thomas Hunt Morgan's BDay

  How Cells Make ATP two fundamental eukaryotic processes
     Autotrophic Metabolism
        Photosynthesis
             Photophosphorylation
chloroplast
     Heterotrophic Metabolism
        Cell Respiration ("Oxidation of Foods")              Aerobic & Anaerobic Respiration
      
      Oxidative Phosphorylation
             Substrate Level Phosphorylation
 
...mainly by phosphorylation of ADP
      





Primary Mechanisms of Phosphorylation
 
 1.
Substrate Level Phosphorylation

                  Pi

     see mcb 12.3*
     steps 7 & 10
     in glycolysis
     
page 482
        or
ecb 13.1
 
 2.
Chemiosomosis (Oxidative Phosphorylation)
                subst-H (glu)   +   NAD+   --->   NADH   +   subst (CO2 + H2O)
                NADH  --e-transport  -->   H+ proton motive force   --ATP synthase-->  ATP
                                                                                                               overview*
 3.
Photosynthetic Phosphorylation
                Light +  H2O  + NADP+    ---->     NADPH   --e--->   H+  --ATP synthase-->  ATP
 

 

 

 

  

Cellular Respiration...   oxidation of food molecules
           Evolution of aerobic metabolism was a major step
           in the history of life on planet Earth...
                         
   
   defined as: series cytoplasmic & mitochondrial...                  
           - linked enzymatic pathways  

          
- catalyze stepwise OXIDATION of food molecules to make ATP     
                  
physiological view:  uptake of O2  &  release of CO
2                    
                   biochemical view
:   O2 consumption  &  CO2 production

 
3 Stages:  1. Digestion of food polymers     -->     monomers   [CH2O]   
                 
2. Production of AcoA  
(via pyruvate)      -->     glycolysis   &/or   FA-oxidation
      

                
 3. Oxidation of AcoA to CO2 & H2O   -->     KC  &  ETC     ecb 13.2 pg 429*
                                                                                  

 

 

 

 

 
  
  4 Pathways of cellular respiration: (glucose oxidation in eukaryotes)
  Glyco-lysis                             [cytoplasm]
         glucose  -->  pyruvate + NADH + ATP
  Krebs Cycle                            [mitochondria]
        Acetyl-coenzymeA  -->  CO2 + NADH + GTP + FADH2
  Electron Transport Chain (ETC)         [mitochondrial membrane]    
        passage of e's from NADH to O2  --->  H2O  +  H+ gradient
  ATP synthase                                                  [mitochondrial membrane]
        mitochondrial membrane protein which
        makes ATP as H+ move into
mitoplasm with their chemical gradient
              overview mcb6e fig 12.5b*                                  (overview Karp fig 5.5*)      

 

 

 

 

 
GLYCO-LYSIS:  Greek -glykos   =  "sweet" + "splitting      Text description - GLYCOLYSIS*text description

Embden,   Meyerhof,   Parnas  Pathway  
 -
10 step enzymatic pathway -                 overview ecb 13.3
          
hexose (C6)   -->   2 PYR (C3)  +  4ATP (2 net)  +  2NADH    

 - anaerobic = no requirement of oxygen 
 - cytoplasmic location  (
? too structured for randomness in aqueous solution ?)

 - energy investment phase (coupled Rx's
             phosphorylation of low energy intermediates to high energy ones

 -   energy capture phase...
  

         
  2 key reactions:                   
        
            -   redox reaction (glyceraldehyde3-PDH)   mcb6e 12.3 steps 6 & 7*
        
            -   substrate level phosphorylation
     
                                   substrate-P  +  ADP  --->   substrate   +  ATP

        Karp fig 5.6 (pathway)µ   &   mcb6e 12.3µ    &    ecb panel 13.1 pg 432

                                                                                             

 

 


GLYCO-LYSIS...   Ancillary Pathways  or how cells use its products

Fates of PYRUVATE...         
      if
anaerobic – 
          
1.   lactic acid respiration  - via lactic acid dehydrogenase 
(mcb 12.5b muscle)*
          2.
  alcoholic fermentation  - via alcohol dehydrogenase       
(mcb 12.5a ethanol)*
      if
aerobic -
          
3.  
Krebs Cycle -pyruvate enters mitochondrial Krebs

       NADH  SHUTTLES          Text description - NADH SHUTTLES*  text description
                       evolutionary advantage...   moves the e's of the cytoplasmic NADH's 
              
        into mitochondrial NADH or FADH2 for use in the ETC...
                                  glycerol-P shuttle
* (Karp fig5.9)    -  skeletal muscle/brain  
                                 
malate shuttle
*  (mcb 12.11)           -  liver, kidney, heart muscle
  

 

 

 

  
  
KEY REACTIONS of GLYCOLYSIS    

     substrate level phosphorylation   (see  steps 7 & 10*) 
     redox reaction involving NAD    
(see  step 6*  & 
ecb panel 13.1 pg 432)
 

Summary of GLYCOLYSIS    (ecb panel 13.1 pg 433*) 

             2 ATP to initiate pathway 
        
      2 substrate level phosphorylations 
        
      makes 2 ATP (net),   2 NADH,   and 2   PYRUVATE 
        
      Fermentations (lactate & alcohol)  & the Shuttles
 

     

 

 

   
  
KREBS CYCLE  [Citric Acid Cycle or Tricarboxylic Acid Cycle 

a cyclical biochemical pathway resulting in aerobic oxidation of cellular fuels, 
such as
CH2O, fatty acids, & amino acids,  while making CO2H2O,  &  ATP

HISTORY  
   
1910's - enzymatic nature learned - dehydrogenases...  (EC 1. - oxidoreductases)
   1930's - substrates identified -  di-COOH's 
                             experiments on minced flight muscle prep's   (NY city pigeons)
   
1937 - Sir Hans Krebs - citrate synthetase...           (condensing enzyme)
                              acetyl-coA [2C]   +  OAA [4C]   --->   citrate [6C]    +    coA-SH 
   1948 - cycle localized within the mitochondria*
   1961 - Peter Mitchell - proposes Chemiosmosis...  [proton (H+) gradient]

  

 

 

 

 
 
Overall reaction: 
           
acetyl-CoA + 3NAD + E-FAD + GDP + P + 2H2O ---->  
                      
                          --->    CoASH + 3NADH + E-FADH2 + GTP + 2CO2

       ENZYMES of KREBS CYCLE     mcb fig 12.10*  &   overview of aerobic respiration*    
           
5  dehydrogenases - ISDH, a KGDH, SDH, MDH, & PDH 
           
2  hydrolyases - aconitatse & fumarase 
           
1  thiokinase - succinyl thiokinase 
           
1  synthetase - citrate synthatase    
           
2  multi-enzyme complexes 
- each with 60 proteins  &  5 coenzymes 
       
                    1.   pyruvate dehydrogenase  (figure*) 
       
                     2.  alpha ketoglutarate dehydrogenase

               µ  but prior to Krebs need to get pyruvate into mitochondria...  figure* 
   

 

 

 

 Pyruvate Dehydrogenase Complex...                                                        
         
catalyzes the Oxidative Decarboxylation of an alpha-Keto acid  (pyruvate)

                          

   3 enzymes : 60 proteins subunits    (figure)*   
   
            A.  pyruvate decarboxylase   
                                
12  dimers = 24 identical subunits 
       
        B.  lipoamide reductase transacetylase (reductase)  
    
                             8  trimers = 24 identical subunits, each 3 lipoates 
   
             C.  dihydrolipoyl dehydrogenase  
   
                                6  dimers 12 subunits with FAD

 

 

 

  PDH COMPLEX Reactions... 
  A. pyruvate decarboxylase    B. lipoamide reductase transacetylase    C. dihydrolipoyl dehydrogenase 
  5 coenzymes
           1. CoASH      (8.8 p309*)               
        
2. Lipoate     (figure)*     
      
  3. Thiamine pyrophosphate  (figure)*
      
  4. E-FAD       (2.26 p54)*   
        
5. NAD+        (2.26 p54)*  
pantothenate (B5)
lipoic acid (antioxidant)
thiamine (B1) 
riboflavin (B2)
niacin (B3)
    Mechanism of Action of PDH Complex*        Text description - KREBS CYCLE*   a text description of PDH

 

 

 

 
the Cycle -  mcb version of cycle - panel 13.2*        pdf version of Krebs - panel 13.2 pg450
                                                                                     a detailed  version by Karp - fig 5.7        
  
    Key Metabolic Reactions of KREBS CYCLE     [+ PDH reaction]
   
                     NAD is reduced         (NADH  -  3/ACoA)
                        substrate level phosphorylation occurs  
GDP + P --> 2 GTP/glu  ( @ ATP)
                        decarboxylation          (
-COOH  -  2x/AcoA)
                        acylation via CoASH    (
succinyl-CoA2x)
     
    thus Each turn of the Cycle                                                        
   
                     4 protons passed to coe's   (3 NADH  &  1 FADH2
                  
     2 CO2's are released                                     
                       
1  GDP is phosphorylated to GTP (equivalent to ATP)
  
                                                  

 

 

 

 
  
Carbohydrates vs. Fats as energy sources ?

    FATTY ACID Metabolism      [ energy sources = fats vs. carbs - fig 13.10* ]        

         oxidation Fatty Acids:  triacylglycerol = mbc p491.Fatty Acids*        
                                       fat/lipid droplet & fatty acid= figure  &   ecb 2.4 pg 72
                    
    µ converts free fatty acids in blood into to Acetyl-CoA
                                       in the mitochondria

   

3 Steps of Fatty Acid Oxidation Cycle   [beta oxidation]                

      
1.   oxidation of COOH end of free fatty acid   &   linking  FFA  to  CoASH
 
      
2.   transport of fatty acyl-coA into mitoplasm from cytoplasm

   
   3.   oxidation of fatty acyl-coA into 2 carbon fragments of Acetyl-CoA 

  

 

 

 

 
 4 enzymes of beta-oxidation cycle...          

1.   fatty acyl-coA LIGASE                  (on outer mito. membranes
                FA-COOH + ATP + CoASH   <-->   FAcoA + AMP + PP 
                
            converts cyotplasmic FA to Fatty-acyl-coA  [c-c-c-c-ScoA]

2.   carnitine acyl-TRANSFERASE 1      (outer mito memb.
        
      FattyCoA + carnitine   <->   Fatty acyl-carnitine + CoASH 
                                 transfers FAcoA to carnitine for transport across mito

3.   carnitine acyl-TRANSFERASE 2       (in mitoplasm)                             
              Fatty acyl-carnitine + CoASH   <-->   FAcoA + carnitine 
                                  releases carnitine & leave FAcoA inside the mitoplasm

4.   fatty acyl-coA DEHYDROGENASE   (in mitoplasm)
         
reduces  FAD and NAD+ in 4 steps

                                                  [my figure*]             [another figure]

 

 

 


 
 
Beta-Oxidation Cycle and the fatty acyl-coA dehydrogenase enzymes
  

           
four steps for this mitochondrial dehydrogenase enzyme system... 
        
             a)  dehydrogenation w FAD --> FADH
                     
b)
 hydration - addition of water 
                     
c)  dehydrogenation - NAD --> NADH. 
                 
     d)  thiol clevage  with CoASH 
                              
mcb 12.12 FA oxidation    &   fig 13.9b pg 438*    &   Karp fig 5.8 pg191
                                
 

    release of a 2carbon fragment as Acetyl-CoA

   Net result:     each turn of the cycle shortens a long chain fatty acid by 2 carbons
                          generating 1
AcoA,  1 NADH and  1 FADH2 for entry into Krebs cycle...
 
  

 

 

 

 

        

             Balance Sheet Aerobic Oxidation   6C- glucose   vs.    6C- FFA

  µ  Rule of Thumb...    the  P/O ratio   (via mito & ETC)       1 NADH  =  3 ATP   &   1 FADH2  =  2 ATP 

Cell RESPIRATION via glucose beta-OXIDATION via 6C-FFA (c-c-c-c-c-c)
 to start - GLYCOLYSIS                         2  ATP  @  LIGASE step       1  ATP
   glyceraldehyde DH
   PGA kinase (
via SLP)
   pyruvate kinase (
via SLP)
  + 2 NADH*   + 6  ATP 
                      + 2  ATP
                      + 2  ATP
 
 KREBS Cycle per each PYR    per 2 cycles @ Fatty-AcoA-dehydrogenase
    PDH                 - 2CO2   + 2 NADH      + 6  ATP          + 3 AcoA* 
         + 2 FADH
2
           
+ 2 NADH 


    +    4  ATP
    +    6  ATP
   
+  10  ATP
    ISDH                - 2CO2 
   
αKGDH           
-  2CO2 
    thiokinase
    SDH 
    MDH
  + 2 NADH      + 6  ATP
  + 2 NADH      + 6  ATP
  + 2 GTP        + 2  ATP
  + 2 FADH2     + 4  ATP
 
+ 2 NADH       + 6  ATP
 Totals 1 glucose --> 2 ATP + 2 pyruvate --->
       - 6 CO2  + 2 ATP  + 10 NADH
*  + 2 FADH2 + 2 GTP
Totals   6C-FFA via   beta-oxidation   -->     10 ATP
                 &
 *Krebs cycle (3 AcoA)  -->  ~ 36 ATP
       *per gly+Krebs+shuttles     total ATP     =  36 to 38
 
                         ATP via 1 AcoA alone  =  12
   total ATP = ~ 46 ATP - 1 ATP   =   ~ 45 ATP
                                  a gain of 
~ 7 to 9 ATP

    

 

 

 

 

 


 
Regulation of Glucose Metabolism & Krebs Cycle...

     substrate availability - mass action controls flow of intermediates [out & in] -  fig 13.23*
    
allosteric inhibition - PFK-1&2*  &  classical feedback inhibition models - fig 4.38 pg 152*
    
covalent modification - reversible phosphorylation... 
   
                                                 protein kinases & phosphoprotein phosphatases     [Review

  µ   4 key enzymes are involved in Krebs regulation… 
        1. PDH           [aresenic poisoning] + SER-P by kinase   - inactive
        2. citrate synthetase + ADP                      - ATP/NADH/cit/AcoA 
        3. isocitrate dehydrogenase + ADP/Ca+2            - ATP 
        4. alpha-keto gluatarate dehydrogenase + Ca+2                    - AcoA   &   NADH

             my figure*                    [apply regulation to the  metabolic charts &  Nicholson ]
 

 

 

 

 

 

 

 

 

 

 

  ENZYME REGULATION…   by adjusting reaction rates of existing enzyme

       via...  COVALENT MODIFICATION of existing enzyme...

                      - addition of
P to an inactive enzyme --> activate enzyme via P transfer

                      - reversible phosphorylation changes protein conformation 

       
              - done by
PROTEIN KINASES,  which transfer P from ATP    fig*
                                       tyrosine kinases add P to TYR residues of enzymes de/activating them
                                  serine/theronine kinases add P to SER or THR residues

                      - PROTEIN PHOSPHATASES...  dephosphorylate, thus inactivating
  

 

 

 

 

 

 

 

 

 

 

 

            the Key metabolic reaction is a   -->    REDOX Reaction...  Text Description - Redox reactions
               AH     +   BO    < --- >     A    +   BOH 
         C6H12O6 +   6O2  < --- >   6CO2 +  6H2O + e's 

 

recall... the 3 steps in the Mitochondrial Oxidation of PYR
                1.  PYR --> CO2 + H2O   -->   NADH/FADH2    Krebs 
   
             2.  e- of NADH/FADH2 -->  O2 to make H2 ETC 
                3.
  ADP + P ---> ATP      H+ transport via Chemiosmosis