# 2 Degradation of Amino Acids

1. Discuss in general terms the use of amino acids for the synthesis of nitrogen-containing compounds 2. Discuss the various functions of glutamine 3. Discuss the roles of transamination 4. Discuss the glutamate dehydrogenase (GDH) & regulation 5. Discuss ammonia disposal

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 NH 4 + from the periphery to the liver for AA catabolism .

Most nitrogenous waste is disposed of as → urea. N is also disposed of as NH 4 + , uric acid & creatinine

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

Transamination Reaction

1. There are many transaminases

2. Coenzyme is pyridoxal PO

from vitamin B6

4

(PPal) formed 3. All AA except THR &v LYS can undergo transamination with α ketoglutarate 4. Equilibrium of reaction is close to 1 therefore reaction direction depends on the [reactants] which are directed by other cellular processes 5. Directionality  removal/addition of products of AA pool 6. Urea Synthesis provides direction by withdrawing amino groups from the AA pool  increase deamination and AA catabolism

Transaminases – Clinical Use

1.

2.

3.

4.

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)

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 NH 4 +

Glutamate Dehydrogenase Coupling

The release of  amino groups as NH 4 + is catalysed by glutamate dehydrogenase through oxidative deamination. Since the reaction is reversible it can also synthesize amino groups.

GDH Requirements

1.

The enzyme is the principal source of NH 4 + 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 (NH 4 + ) can be fixed to a C skeleton. Therefore essential AA could be provided in the diet as because NH 4 +   keto acids and the amino groups as NH 4 + glutamate  other AA by transamination.

(iii)

GDH is the major AA oxidative pathway and the major source of NH 4 + Also provides directionality to transamination/GDH. In vivo,  [GLU] , NAD + & removal of NH 4 + drive deamination of glutamate. With excess NH 4 + (bacterial metabolism in intestine), glutamate can be formed.

Glutamate Dehydrogenase

1. Driving Force: necessity to maintain low levels of ammonia which is toxic. Therefore Transaminase + GDH mediates α amine  NH 3  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

Regulation of GDH

Regulation of GDH: allosteric control through diverse substances. Major: (i) energy  (ii) AA load  is there enough? If not oxidize AA 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  c)  NAD H inhibits GDH TCA cycle to produce ATP/GTP

AA LOAD:

Excess AA: override inhibition caused with  themselves can  GDH activity.

energy therefore AA

Ammonia Disposal

NH 4 + 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 + H 2 O  GLU  GLU + NH glucose/energy 3 + H + (glutaminase) NH 3 (diffuses across tubular membranes) + H + (urine)  NH 4 + This lowers [H + ] and increases H + /Na + exchange Accounts for 1/2 to 2/3 of daily acid load

Ammonia Disposal via Kidney

Chronic Metabolic Acidosis

Activation of NH 4 + excretion system takes 2-3 days and is maximal at 5-6 days.  glutaminase, GDH, mitochondrial glutamine transport  NH 4 + urinary excretion   renal gluconeogenesis from AAs urea synthesis by liver (therefore more GLN available to kidney)

Importance of NH

4 +

in acidosis

(i) pKa of NH 4 = 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 NH 4 + readily available from AA) (NH 3 (iii) spares body stores Na + and K + which are excreted with titratable acids (H 2 PO 4 ) to balance +/- (maintain electrical neutrality). Na + used first, then K + therefore NH 4 + availability important, since it can fulfill this function.

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  NH 4 + disposal

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%.

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

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)

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

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