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Amino Acids Catabolism Nitrogen atoms catabolism Transamination reactions (aminotransferases, transaminases) Oxidative (NAD an FAD dependent) & non-oxidative deamination reactions - NH3 production and Transport or Ammonia Assimilation a.Glutamate dehydrogenate b.Glutamine synthetase c.Carbamoyl phosphate synthetase-I Urea cycle Carbon skeletons catabolism Amino Acids Anabolism Amino Acids Biosynthesis
ENVIRONMENT
ORGANISM
Ingested protein
1
Biosynthesis 2 3
Protein
a AMINO ACIDS c
Degradatio n (required) Nitrogen (ketogenic)
b c
Purines Pyrimidines Porphyrins
Carbon skeletons (glucogenic) Used for energy pyruvate -ketoglutarate succinyl-CoA fumarate oxaloacetate
Urea
acetoacetate acetyl CoA
Amino acids are grouped into 2 classes, based on whether or not their carbon skeletons can be converted to glucose: glucogenic ketogenic. Carbon skeletons of glucogenic amino acids are degraded to:
pyruvate, or a 4-C or 5-C intermediate of Krebs Cycle. These are precursors for gluconeogenesis.
Glucogenic amino acids are the major carbon source for gluconeogenesis when glucose levels are low. They can also be catabolized for energy or converted to glycogen or fatty acids for energy storage. Carbon skeletons of ketogenic amino acids are degraded to: acetyl-CoA, or acetoacetate. Acetyl CoA, & its precursor acetoacetate, cannot yield net production of oxaloacetate, the gluconeogenesis precursor. For every 2-C acetyl residue entering Krebs Cycle, 2 C leave as CO2.
Carbon skeletons of ketogenic amino acids can be catabolized for energy in Krebs Cycle, or converted to ketone bodies or fatty acids. They cannot be converted to glucose.
The 3-C a-keto acid pyruvate is produced from alanine, cysteine, glycine, serine, & threonine. Alanine deamination via Transaminase directly yields pyruvate. Serine is deaminated to pyruvate via Serine Dehydratase. Glycine, which is also product of threonine catabolism, is converted to serine by a reaction involving tetrahydrofolate (to be discussed later). The 4-C Krebs Cycle intermediate oxaloacetate is produced from aspartate & asparagine. Aspartate transamination yields oxaloacetate. Aspartate is also converted to fumarate in Urea Cycle. Fumarate is converted to oxaloacetate in Krebs cycle. Asparagine loses the amino group from its R-group by hydrolysis catalyzed by Asparaginase. This yields aspartate, which can be converted to oxaloacetate, e.g., by transamination. The 4-C Krebs Cycle intermediate succinyl-CoA is produced from isoleucine, valine, & methionine. Propionyl-CoA, an intermediate on these pathways, is also a product of b-oxidation of fatty acids with an odd number of C atoms. The 5-C Krebs Cycle intermediate a-ketoglutarate is produced from arginine, glutamate, glutamine, histidine, & proline. Glutamate deamination via Transaminase directly yields a-ketoglutarate.
Glutamate deamination by Glutamate Dehydrogenase also directly yields a-ketoglutarate. Histidine is first converted to glutamate. The last step in this pathway involves the cofactor tetrahydrofolate. Tetrahydrofolate (THF), which has a pteridine ring, is a reduced form of the B vitamin folate. Within a cell, THF has an attached chain of several glutamate residues, linked to one another by isopeptide bonds involving the R-group carboxyl.
Nitrogen Balance
Nitrogen balance = nitrogen ingested - nitrogen excreted (primarily as protein) (primarily as urea) Nitrogen balance = 0 (nitrogen equilibrium) protein synthesis = protein degradation Positive nitrogen balance protein synthesis > protein degradation Negative nitrogen balance protein synthesis < protein degradation
Amino acid metabolism is intimately intertwined with nitrogen acquisition All organisms need bio-available sources of
N2 is converted to metabolically useful forms (is "fixed") only by a few species of prokaryotes, called diazatrophs. Diazatrophs of the genus Rhizobium live symbiotically in the root nodules of legumes, where they convert N2 to NH3 (ammonia) in a process called NITROGEN FIXATION
NITROGENASE
* But, less than 1% of N entering the biosphere comes from N fixation. Another oxidized form of nitrogen, NO3 - (nitrate ion) is also found in the soils and oceans. It is converted to NH4 + through... NITRATE ASSIMILATION:
* The reduction of NO3- to NH4 + (ammonium ion) occurs in green plants, various fungi, and certain bacteria in a two-step pathway:
(1) The 2-electron reduction of nitrate to nitrite: NO3- + 2 H+ + 2 e- NO2- + H2O ( catalyzed by nitrate reductase) (2) This is followed by the 6-electron reduction of nitrite to ammonium: NO2- + 8 H+ + 6 e- NH4+ + 2 H2O ( catalyzed by nitrite reductase)
*NH3/NH4+ can be incorporated into the amino acids glutamate by glutamate dehydrogenase and glutamine by glutamine synthetase.
No animals are capable of either N-fixation or nitrate assimilation, so they (we!) are totally dependent on plants and microorganisms for the synthesis of organic nitrogenous compounds, such as amino acids and proteins, to provide this essential nutrient.
stomach
pepsin
elastase
Excretion
Nitrogen is also constantly being lost from the body in a variety of different forms. The principal excretory nitrogen product in mammals is urea (about 12-20 g urea nitrogen/day). *Other excretory products include ammonium ion (fish), uric acid (birds & insects) and creatinine (human). The amounts of each of these is dependent of the metabolic state of the individual. Some animals switch from one form to another during development (more about that later)
TRANSAMINATION REACTIONS
Amino Acids alpha ketoglutarat NH3 CO2
Alphaketo acid
L-Glutamat
Urea
TRANSAMINASE ENZYMES
1. Alanin transferase (alanin-pyruvate transaminase) 2. Glutamate transaminase (glutamate- -ketoglutarate transaminase) COENZYME : Pyridoxal Phosphate (PLP) Transaminases equilibrate amino groups among available a-keto acids. This permits synthesis of non-essential amino acids, using amino groups from other amino acids & carbon skeletons synthesized in a cell.
Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized.
Although the amino N of one amino acid can be used to synthesize another amino acid, N must be obtained in the diet as amino acids (proteins)
The usual AA acceptor is -ketoglutarate, producing GLUTAMATE (GLU) and the new a-keto acid. What is produced if oxaloacetate is the acceptor?
What is produced if oxaloacetate is the acceptor? It is one C shorter than a-ketoglutarate, so produces..
X
oxaloacetate
X ASPARTATE
The second typical step in AA deamination involves transfer of the amino group from Glutamate to oxaloacetate, yeilding -ketoglutarate and Aspartate (ASP)
glutamate-aspartate aminotransferase
Deamination reactions
Amino group (and H) removed
Forms ammonia (NH3) Carbon skeleton left can be
Oxidized in Krebs Cycle used for gluconeogenesis converted to fatty acid a. Oxidative deamination
In order to regenerate the amino acceptor, -ketoglutarate and to provide ammonia for re-utilization or disposal, it is necessary to deaminate glutamate. This is accomplished by the action of the enzyme glutamate dehydrogenase (GDH) which is located in the mitochondrial matrix.
Glutamate Dehydrogenase catalyzes a major reaction that effects net removal of N from the amino acid pool.
OOC
H2 C
H2 C
glutamate
H2O
O H2 H2 OOC C C a-ketoglutarate C
Glutamate Dehydrogenase
It is one of the few enzymes that can use NAD+ or NADP+ as e- acceptor.
Amino acid
a-ketoglutarate
NADH + NH4
a-keto acid
glutamate
NAD + H2O
Transaminase
Glutamate Dehydrogenase
Summarized above: The role of transaminases in funneling amino N to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH4+.
b. Non-Oxidative deamination
Oxidative deamination is the most active mechanism by which animal cells remove nitrogen from their amino acids. However, the mammalian cells carry out non-oxidative deamination reactions with the enzymes glutaminase, asparginase and histidinase, all of which yield ammonia as a product. Glutaminase is probably one of the most prominent. The action of glutaminase in the liver yields glutamic acid and ammonia, the latter of which is consumed by the urea cycle.
Carbon skeletons are used for energy. Glucogenic: TCA cycle intermediates or pyruvate (gluconeogensis)
Urea cycle
Detoxification of Ammonia by the Liver Liver very effective at eliminating ammonia from blood
Ammonia is toxic
Readily ionizes to ammonium ion NH4+
NH4+ converted to urea in liver (urea cycle)
Urea contains 2 x NH2
aspartate
Urea excreted in urine
UREA CYCLE
mitochondri a cytosol
UREA FORMATION
UREA CYCLE :
O H 2N C NH2
urea
Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it. Urea is less toxic than ammonia. The Urea Cycle occurs mainly in liver.
The 2 nitrogen atoms of urea enter the Urea Cycle as NH3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate. The NH3 and HCO3- (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate.
HCO 3-
Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with carbonyl phosphate and carbamate intermediates. Ammonia is the N input.
carbonyl phosphate
O O-
carbamate
carbamoyl phosphate
Alternate forms of Carbamoyl Phosphate Synthase (Types II & III) initially generate ammonia by hydrolysis of glutamine. The type II enzyme includes a long internal tunnel through which ammonia & reaction intermediates such as carbamate pass from one active site to another.
HCO 3ATP ADP O HO NH3 Pi H2N ATP ADP O H2N C OPO32C C OPO32-
carbonyl phosphate
O O-
carbamate
carbamoyl phosphate
O H2N C
carbamoyl phosphate
Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation.
glutamate (Glu)
H H3N+ C CH2 CH2 COOCOO-
N-acetylglutamate
O H3C C N H H C CH2 CH2 COOCOO-
Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric activator N-acetylglutamate. This derivative of glutamate is synthesized from acetyl-CoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.
O OPO32-
C NH CH2
NH2
Urea Cycle
Enzymes in mitochondria: 1. Ornithine Transcarbamylase Enzymes in cytosol: 2. ArgininoSuccinate Synthase 3. Argininosuccinase 4. Arginase.
H2N
H2N
carbamoyl phosphate 1
Pi
CH2 CH2 HC
citrulline
COOCH2 HC NH2
COO
Urea Cycle
4
NH3+
ornithine
O C NH2 H2N C NH CH2 H2O NH2+
2
COO
-
COO-
aspartate
C NH CH2 CH2 CH2 HC NH3+ NH2+
urea
CH2
3
COOHC CH NH3+ COO-
HC
H N
COO-
arginine
CH2 CH2
argininosuccinate
HC
COO-
fumarate
COO-
cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine urea arginine fumarate citrulline aspartate argininosuccinate
For each cycle, citrulline must leave the mitochondria, and ornithine must enter the mitochondrial matrix. An ornithine/citrulline transporter in the inner mitochondrial membrane facilitates transmembrane fluxes of citrulline & ornithine.
cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine urea arginine fumarate citrulline aspartate argininosuccinate
A complete Krebs Cycle functions only within mitochondria. But cytosolic isozymes of some Krebs Cycle enzymes are involved in regenerating aspartate from fumarate.
COOCH2 CH2
HC
NH3+
COO-
COO-
COO-
COO-
Aminotransferase (Transaminase)
Fumarate is converted to oxaloacetate via Krebs Cycle enzymes Fumarase & Malate Dehydrogenase. Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate). Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.
Treatment Stop protein intake Dialysis Increase ammonia excretion: Na benzoate, Na phenylbutyrate, L-arginine, L-citrulline Liver transplantation
44
Postulated mechanisms for toxicity of high [ammonia]: 1. High [NH3] would drive Glutamine Synthase: glutamate + ATP + NH3 glutamine + ADP + Pi This would deplete glutamate a neurotransmitter & precursor for synthesis of the neurotransmitter GABA. 2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ a-ketoglutarate + NAD(P)H + NH4+ The resulting depletion of a-ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.
cytosol
ornithine citrulline However some urea aspartate enzymes of the arginine argininosuccinate pathway are in fumarate other cells and tissues where they generate arginine & ornithine, which are precursors for other important molecules. E.g., Argininosuccinate Synthase, which catalyzes synthesis of the precursor to arginine, is in most tissues. Mitochondrial Arginase II, distinct from the cytosolic Urea Cycle Arginase, cleaves arginine to yield ornithine.
arginine (Arg)
H H3N+ C CH2 CH2 CH2 NH
COO-
O O-
NH2+
creatine
C NH2
NH2
The amino acid arginine, in addition to being a constituent of proteins and an intermediate of the Urea Cycle, is precursor for synthesis of creatine & the signal molecule nitric oxide.
+ H2 N=C-HNCH2 CO 2 Guanidoacetate
SAM + ATP
NH2
Arginine
H N
Glycine
Ornithine
Creatinine (Urine)
HN N CH3
S-Adenosylhomocysteine + ADP
Creatine
+ H2 N=C-NCH2 CO 2 CH3
Phosphocreatine
48
NHPO3-2
ATP
ADP + Pi
49
NH2
NH3+
NO
Citrulline
50
Nitric Oxide
Cell messenger Implicated in a wide range of physiological and pathophysiological events: Vasodilation: Activates guanylyl cyclase cGMP Nitroglycerin Glycerin + NO Sildenafil (Viagra): in vascular smooth muscle:
Blocks
Phosphodiesterase-5
NO
cGMP
GMP
51
NO is a short-lived signal molecule with diverse roles in different cell types, including regulation of smooth muscle contraction, gene transcription, metabolism, and neurotransmission. Many of the regulatory effects of NO arise from its activation of a soluble cytosolic Guanylate Cyclase enzyme that catalyzes synthesis of cyclic-GMP (analogous in structure to cyclic-AMP). Cytotoxic effects of NO observed under some conditions are attributed to its non-enzymatic reaction with superoxide (O2-) to form the strong oxidant peroxynitrite (ONOO-).
COO-
There is no tRNA for citrulline & this amino acid is not incorporated translationally into proteins.
C O
NH2
arginine
citrulline
However, Ca++-activated Peptidylarginine Deiminases convert arginine residues within proteins to citrulline as a post-translational modification.
GABA Formation
NH3+
-
Glutamate decarboxylase
NH3+
-
CO2
GABA is an important inhibitory neurotransmitter in the brain Drugs (e.g., benzodiazepines) that enhance the effects of GABA are useful in treating epilepsy
54
CAC intermediates are: 1. Diverted to gluconeogenesis (formation of glucose) 2. Diverted to ketogenesis (formation of ketone bodies) 3. Completely oxidized to CO2 and H2O
*
Ketone Bodies or Fatty Acids
* *Glucogenic*
Ketogenic
Glucose
* *
Figure 18-14, 18-29
Biochemistry 221 - Metabolism
Regulation of amino acid and protein metabolism -demand for amino acid and protein -demand for amino acid and protein anabolism -eg if need positive N anabolism -eg if need positive N balance or to keep in N equilibrium balance to keep in N equilibrium -if lots of glucoseor available
-demand for amino acid and protein catabolism-eg if in negative N balance or to keep in N equilibrium
-demand for amino acid and protein catabolism-eg if in negative N balance or to keep in N equilibrium
Phenylketonuria: phenylpyruvate and Phe accumulate in blood & are excreted in urine One of the first inheritable disease discovered in humans! The disease can be controlled by a strict diet - avoiding slowed mental development