1. # 2 Degradation of Amino Acids
Discuss in general terms the use of amino acids for the synthesis of nitrogen-containing compounds
Discuss the various functions of glutamine
Discuss the roles of transamination
Discuss the glutamate dehydrogenase (GDH) & regulation
Discuss ammonia disposal

2. Amino Acid Degradation
Degradation : → removal & disposal of amino group
→ utilization of the carbon skeleton for energy and gluconeogenesis
ALA & GLN → non-toxic vehicles for transport of NH4+ from the periphery to the liver for AA catabolism
Most nitrogenous waste is disposed of as → urea. N is also disposed of as NH4+, uric acid & creatinine .

3. Transamination Transamination
15 N AA  free exchange among AA (except THR & LYS)
(not true of the carbon portion)
Enzyme = transaminase or aminotransferase
Quantitatively most important reaction of AA metabolism
Involved in:
Synthesis NEAA
Degradation most AA Redistribution

4. Transamination Reaction
There are many transaminases
Coenzyme is pyridoxal PO4 (PPal) formed from vitamin B6
All AA except THR &v LYS can undergo transamination with α ketoglutarate
Equilibrium of reaction is close to 1 therefore reaction direction depends on the [reactants] which are directed by other cellular processes
Directionality  removal/addition of products of AA pool
Urea Synthesis provides direction by withdrawing amino groups from the AA pool  increase deamination and AA catabolism

5. Transaminases – Clinical Use

6. Transaminases – Clinical Use
ASP & ALA transaminases are the most abundant
Several are present in both cytosol and mitochondria (isoenzymes)
ASP aminotransferase is one of the most frequently assayed enzymes in the clinical laboratory. Its determination in serum  diagnostic acid especially for assessing liver disorders
Nomenclature of transaminases is confusing: same enzyme =
aspartate – glutamate transaminase
aspartate transaminase
glutamate – oxaloacetate transaminase
SGOT (clinical literature)

7. Role of Transamination
(i) Redistridution of  amino groups to balance AA pool
-- dietary proteins provide a mixture of AA whose proportions differ from AA pool required by body
 correct imbalance
(ii) AA synthesis / degradation performed in conjunction with glutamate dehydrogenase (GDH)
GDH can remove or add amino groups to the AA pool
Most  amino groups  glutamate due to the action of transaminases.
When there is a surplus of AAs in the pool, the  amino groups can be funneled through glutamate and released as NH4+

8. Glutamate Dehydrogenase Coupling
The release of  amino groups as NH4+ is catalysed by glutamate dehydrogenase through oxidative deamination. Since the reaction is reversible it can also synthesize amino groups.

9. GDH Requirements
1. The enzyme is the principal source of NH4+ in the body. GDH is a mitochondrial enzyme located in matrix, present in liver cells and most tissues.
2. Important for three reasons:
(i) Link between TCA cycle & metabolism of AA ( keto acids are TCA cycle intermediates).
(ii) In mammals, only reaction in which an inorganic molecule (NH4+) can be fixed to a C skeleton. Therefore essential AA could be provided in the diet as  keto acids and the amino groups as NH4+ because NH4+  glutamate  other AA by transamination.
(iii) GDH is the major AA oxidative pathway and the major source of NH4+
Also provides directionality to transamination/GDH. In vivo,  [GLU] , NAD+ & removal of NH4+ drive deamination of glutamate. With excess NH4+ (bacterial metabolism in intestine), glutamate can be formed.

10. Glutamate Dehydrogenase
1. Driving Force: necessity to maintain low levels of ammonia which is toxic. Therefore Transaminase + GDH mediates α amine  NH3  urea
2. Glutamate: link between transamination and Urea synthesis
Transamination  funnels amino groups through glutamate & a single dehydogenase suffices therefore activity of GDH is key

11. Regulation of GDH (i) energy  is there enough? If not oxidize AA
Regulation of GDH: allosteric control through diverse substances. Major:
(i) energy  is there enough? If not oxidize AA
(ii) AA load  surplus? Therefore degrade (even when energy is high)
Energy:
a) GTP (&  ATP) inhibit GDH. When GTP (TCA cycle) & ATP (glycolysis / oxid. phosphorylation) are , energy index cell  therefore GDH 
b) Conversely ADP and GDP , energy  therefore GDH active in order to produce Keto acids  TCA cycle to produce ATP/GTP
c)  NAD H inhibits GDH
AA LOAD:
Excess AA: override inhibition caused with  energy therefore AA themselves can  GDH activity.

12. Ammonia Disposal NH4+ must be disposed of due to toxicity
symptoms = feeding intolerance, vomiting, lethargy. Irritability, respiratory distress, seizures and coma
Most is disposed of through urea (to be discussed)
Smaller amounts can be disposed of through the kidney via GLN
GLN + H2O  GLU + NH3 + H (glutaminase)
GLU  glucose/energy
NH3 (diffuses across tubular membranes) + H+  NH4+ (urine)
This lowers [H+] and increases H+/Na+ exchange
Accounts for 1/2 to 2/3 of daily acid load

13. Ammonia Disposal via Kidney

14. Chronic Metabolic Acidosis
Activation of NH4+ excretion system takes 2-3 days and is maximal at 5-6 days.
 glutaminase, GDH, mitochondrial glutamine transport
 NH4+ urinary excretion
 renal gluconeogenesis from AAs
 urea synthesis by liver (therefore more GLN available to kidney)

15. Importance of NH4+ in acidosis
(i) pKa of NH4 = 9.3
(therefore does not lower pH of urine)
pH must be  4.4 since Na+ / H+ exchange cannot function if the difference is > 1000 fold (pH 7.4/4.4 = log3 = 1000)
(ii) large amounts of acids can be excreted as NH4+ (NH3 readily available from AA)
(iii) spares body stores Na+ and K + which are excreted with titratable acids (H2PO4) to balance +/- (maintain electrical neutrality). Na+ used first, then K+ therefore NH4+ availability important, since it can fulfill this function.

16. High Protein Diets PRO Reduce fat intake
Less calorie dense than fat, may reduce total caloric intake
Important for athletes who have  P breakdown (exercise)
But  P diet = animal P =  fat as well
ANTI
Excess over caloric needs will be converted to glucose/glycogen
AA metabolized, fat stores untouched
Increased liver & renal stress due to  NH4+ disposal

17. Case #2 Amino Acid Degradation: Starvation Case Discussion
An obese patient volunteered to go on a starvation diet as part of a study of amino acid metabolism. Blood samples were taken and analyzed for plasma amino acids for as long as 5 to 6 weeks following the fast. Blood ketone bodies increased at the end of the first week.
Valine, leucine, isoleucine, methionine, and α amino-butyrate concentrations were transiently increased during the first week, but dropped below initial levels later. Glycine, threonine, and serine levels decreased more slowly.
13 other amino acids eventually decreased. The decrease was largest for alanine, which dropped 70% in the first week. Total plasma amino nitrogen concentration decreased only 12%.

18. 1. What changes in carbohydrate, lipid & protein metabolism occur at the beginning of a fast?
Body’s major energy source: fat & glucose (not protein)
Beginning fast: muscle → switches FA oxidation
brain → needs glucose
liver → breakdown glycogen → glucose
blood → maintain glucose
Long term fast: brain → ketone bodies use
liver → ketone bodies from FA
In between… liver must maintain glucose therefore uses AA → glucose
Muscle + Liver → Protein breakdown → AA

19. 2. Explain the ketosis and acidosis observed in starvation.
KETOSIS: FA oxidation  acetyl CoA  ketone (Brain)
ACIDOSIS: ketones  pH blood, normally buffered with bicarbonate which , when maxed hyperventilation then  CO2.
3. What might cause an increase in plasma branched-chain amino acids after 5 days of starvation?
BCAA , from MUSCLE & LIVER protein breakdown, associated with starvation, diabetes (imbalances)

20. 4. Why do the other amino acids eventually decrease?
Because of depletion of protein « reserves », below this level, use would compromise cellular function
5. Why does total plasma amino nitrogen only decrase by 12%?
As catabolism (use) of AA → glucose ↑, protein breakdown ↑ to compensate, maintaining AA constant

21. 6. Is the decreased plasma alanine concentration related to gluconeogenesis? What is the alanine-glucose cycle? Why does alanine drop so much?
Pyruvate  ALA transport from muscle  liver for glucose synthesis. When P breakdown is maxed, then ALA decreases