September 25, 1997                    MAST 634                    Lecture 7: Fermentation 

Major Points from Last Lecture 
      Uses of fluorescence 
            inverse (sort of) relationship between fluorescence and photochemistry 
      Overview of Metabolism 
            all lead to central metabolism 
      Calculating theoretical energy yields 
      ATP production: substrate - level versus oxidative ( and photo-) phosphorylations 

Fermentation 
      recycles NAD⁺ 
      NADH oxidization releases energy as heat; in oxidative phosphorylation, it drives PMF that in turn drives ATP synthesis 

End products of glycolysis: pyruvates + 2 ATP + 2 NADH; see handout 

Without O₂: Fermentation oxidizes NADH to NAD⁺ 
Ability to deal with anoxia (no O₂) ancient trait world anoxia for first 10^a years 
      oxygen ---> free radicals (e.g. O₂^-) and perioxide (H₂O₂) 

Miscellaneous Notes about Lactate and EtOH Fermentation 

1. EtOH fermentation 
            Not a single step reaction 

                                                          ADH 
    pyruvate ----> acetaldehyde + CO₂ ----> ethanol 

[see page 466 in V&V] 

2. Lactate fermentation very common 
    Lactic acid bacteria do only this 
          Homofermentation ---> only lactate 
          heterofermentation ---> lactate +EtOH + CO₂ 

These bacteria need very complex media with many compounds 

Trademarks of fermentations 
     1. Starting organic material partially oxidized 
     2. Little or no CO₂ released; organic C is released 
     3. Modest ATP yield, e.g. one or two ATP/reaction 

Anaerobes: living without O₂ 
      Generally bacteria 
      Some eukaryotes, but few 
      Interesting eukaryote: protozoan pathogen, Giardia 
          1. Has no mitochondria; appears to have lost them during evolution 
          2. ATP produced by glycolysis and mixed acid fermentation, i.e. several acids are produced. 

How do cells and animals normally living with O₂ deal with anoxia? 

First, consider cells: 
      Different muscle cells, different levels of glycolysis, etc (substrate - level phosphorylation enzymes) vs. Respiration (oxidative phosphorylation) 

V&V has bird examples 
Let's consider fish examples 

Two basic muscle types 
      1. Red - slow twitch  Sustained E production 
             Red color from hemes (Fe) in mitochondria 
      2. White - fast twitch        short bursts 

High amounts of glycolytic and fermentation enzymes in white muscles. Consider tuna: 40 mph for ~ 10 min with 30 sec between bursts. 

High amounts of LDH in tuna white muscle 
      LDH = lactate dehydrogenase 

But when you go into training, you don't increase red muscle ---> enzyme levels change 

Controls on LDH activity 

      LDH stimulated by anoxic conditions 
      LDH inhibited by oxic conditions 
 
 

	O2	pH	[pyruvate]	creatine -P
oxic	+	~7	low	high
anoxic	-	low	high	low

 

what causes low pH in anoxia? 
      Glucose ---> lactate + 2H⁺ 

But not real reason, according to Hochachka and Somero 

Real reason: ATP hydrolysis 
      ATP + H₂O ---> ADP + P_i + H⁺ 

Aerobic respiration re-uses H⁺ 

Cells with high LDH activity have ability to withstand low pH (=buffering capacity) 

            Deep-sea fish: float and wait, low LDH activity, low buffering capacity 

            Warm-bodied fish: active, high LDH activity, high buffering capacity 

Whole animals withstanding anoxia 
      Important in marine environments ---> sediments 
      Examples 
            parasitic helminths 
            burrowing annelids ("worm") 
            Intertidal bivalves (at low tide) 
            Some vertebrates 
                    Goldfish can survive without O₂ for several days 
                    Freshwater turtles 

Strategies 

       I. Storage of C: glycogen 
              important because anaerobic metabolism yields less ATP 
             Also true in white muscles of aerobic organisms 

           Examples: 
              anoxia - tolerant goldfish 1300 mu  mol gluc./gm 
              anoxia - intolerant trout 235 mu  mol gluc./gm 
     Mytilus: 50% of dry wt can be glycogen! 

What is glycogen? 
      See p485 in V&V 

1. Alph (1 --> 4) glucose polymer 
2. branches 1-6 every 8-12 glucoses 
3. occurs as granules intra cellular (very large) 

Why glycogen and not fat in muscles? 
      1. Muscles degrade glycogen faster 
      2. Fatty - acids (FA) can't be degraded anaerobically --> needs O₂ 
            - O₂ not directly involved 
            - O₂ needed for NADH and FADH₂ oxidation 

oxidation 
            Fa_n - acetyl CoA + NAD⁺ + acetyl-CoA ---> Fa_n-2 - acetyl CoA + NADH 
see page 669 in V&V 

3. V&V says animals cannot convert fatty acids to glucose (and eventually to riboses for nucleic acids) 
     Glucose synthesis: need when animal is "living" on protein (meat or fasting!) 
            = gluconeogenesis 

                              phosphorylase 
     glycogen_N + P_i ----------------> glycogen_n-1 + glucose -1-P 

     G-1-P then enters glycolysis 
     First of many examples where "starting point" for glycolysis is not glucose 

II. Switch to more efficient fermentation in Mytilus 

     In Mytilus, for example 
 

	#ATP's	Survival without O2
lactate	2	3 days
propionate	6	9

 

Other important fermentations 

In cephalopods: Squid can swim very rapidly uses protein and amino acids as energy source 
specifically: arginine~P (<----- arginine +ATP) 

Arg~P ---> ATP + Arg 
    But what to do with Arg 
Arg is very basic ---> osmotic problems 
Arg + pyruvate ----------> octopine; octopine is a 9C amino acid 
NADH -----> NAD⁺ 
octopine is good for osmotic balance 

Use of amino acids (actually, proteins that is hydrolyzed to amino acids) 
 
 

                 Leu ------------------> Keto isocaproate 

Aerobic degradation 

      AA ------> alpha - keto acid ---> Kreb cycle + NH₄⁺ 

III. Reduce metabolic rate 
           much better than switching fermentation 
    e.g. Decreasing metabolism in Mytilus survival goes from 3 to 60 days 
    Effects ATP turnover --but ATP conc. decrease little 
 

	% Aerobic Rate	LT50
Locust	27	72h
Mytilus	5	35 d
Artemia	<5	>24 months

 

Other effects on metabolism (Hand and Hardewig 1996) 

Protein synthesis decreases 
1. Some evidence that transcription is not affected 
2. But genes for some proteins regulated "up regulated", i.e. they are induced 
3. Translation level control? 

Protein turnover 
     both synthesis and degradation of protein 
Generally low in prokaryotes: "unwanted" proteins diluted out as cells continue to divide 

Important in eukaryotes 
      Big decrease in protein degradation with switch to anaerobic metabolism 
      Remaining degradation shifts to ATP independent hydrolysis 

How is protein degradation slowed down? 
      1. Not by directly inhibiting proteases 
      2. Fewer proteins tagged by ubiquitin; ubiquitin is a protein. See pages 101-1015 
         Several ubiquitins covalently link to doomed protein 

In eukaryotes, intracellular hydrolysis of selected proteins carried out y proteosome, structure similar in size (26S) and complexity as ribosomes. 

        protein -- ubiquitin +ATP -------> peptides 
                                           proteosome 

Half-life of proteins set by structure 

          PEST proteins very labile 
                    P Pro 
                    E Glu 
                    S Ser 
                    T Th 
      
      applies to prokaryotes too!