Nitrogen Metabolism Flashcards
(113 cards)
1
Q
protein turnover
A
Proteins are constantly synthesized and degraded and this process is known as turnover. The turnover of proteins occurs at different rates. Some proteins such as collagen have a very long half life some have turnover in few minutes. &5% of amino acids are reutilized for protein synthesis. 25% are used for gluconeogenesis especially during starving
2
Q
nitrogen balance
A
regardless of the nutritional or metabolic status, humans constantly excrete nitrogen. Nitrogen balance is comparison between intake of nitrogen and excretion. Negative nitrogen balace results when dietary protein intake is inadequate. Positive nitrogen balacen is when there is net increase in body proteins
3
Q
what causes positive nitrogen balance
A
growth, pregnancy lactation
recovery from metabolic stress or injury
4
Q
what causes negative nitrogen balance?
A
inadequate dietary protein
metabolic stress, sepsis, trauma
deficiency of an essential amino acid
5
Q
preotein requirement of an individual is determined by what?
A
age pregnancy lactation convalescence after illness catabolic states
6
Q
what factors affect protein utilization
A
quality of protein
digestibility
caloric level of diet
7
Q
what are some of the effects of protein excess?
A
loss of calcium in the urine -> may lead to osteoporosis in women
increase in workload of kidney -> hyperfiltration.
8
Q
What are some of the effects of protein deficiency
A
lack of growth, negative nitrogen balance, reduced serum albumin, edema, increased susceptibility to infection
Marasmic -> ematicated
Kwashikor -> edema and hepatomegaly
relative protein deficiency caused by trauma and sepsis which require the use of proteins for recovery.
9
Q
what are endopeptidases and exopeptidases
A
cleave protein by hydrolyzing peptide bonds within polypeptide chain. Exopeptidases cleave amino acids from either the n or c terminal ends of peptides and proteins
10
Q
what are the ssecretions that facilitate digestion?
A
aqueous - varying pH to provide the optimal environment for the enzymes
enzyme precursors- proteolytic enzymes - inactive precursors, activated after secretion into lumen by limited proteolysis
mucus - lubricant
11
Q
what are the concents of gastric juice
A
HCl (parietal cells)
Gastrin
Pepsinogen
12
Q
Functions of gastrin
A
in response to vagal stimulation
stimulates HCl secretion in parietal cells
pepsinogen secretion stimulation in chief cells
13
Q
purpose of hcl
A
decreases pH
denatures dietary protein
proper pH for pepsin
initiates limited proteolysis of pepsinogen to pepsin
14
Q
activation of pepsinogen
A
process initiated by H+ and becomes autocatalytic. cleave peptide bond. at pH <2, peptide dissociates to give active form of pepsin. in parietal cell deficiency, dissociation does not occur because HCl is insufficient
15
Q
specificity of pepsin
A
broad specificitycleaves to the c terminal side of aromativ and bulky aliphatic amino acid residues to produce large peptide fragments
16
Q
Hormones of duodenum and functions
A
secretin binds to pancreatic cells and stimulates release of pancreatic juice which is slightly alkaline (enriched in HCO3-) to neutralize acidic stomach contents.
CCK two sites of action - binds to exocrine cells of hte pancreas and stimulates the relase of pancreatic zymogens into the lumen of hte small intestine. inactive precursor forms of trypsin, chymotrypsin, elastase, carboxypeptidases A and B enter the intestine through the pancreatic cuct CCK-PZ also acts on gallbladder to initiate contraction and release bile into the lumen of intestines
17
Q
activation of pancreatic zymogens
A
enteropeptidase - brush border (intestinal cells)
catalyzes conversion of trypsinogen to trypsin, initiating cascade of proteolytic events which result in activation of all the pancreatic zymogens
trypsin catalyzes limited proteolysis of all of the remaining zymogens to produce active forms of chymotrypsin, elastase, CPA and CPB
18
Q
Specificity of proteases
A
- each protease has a different specificity and the products of one can be used as substrates for another. Specificities complement one another in such a way that collectively they can disassemble a protein into a mizture that contains about 35% neutral and basic amino acids and 65% oligopeptides
19
Q
What are the endopeptidases
A
Trypsin - cleaves to the C-side of basic amino acids (lys, arg)
Chymotrypsin - cleaves to the C-side of aromatic amino acids (phe, tyr, trp)
Elastase - cleaves to the C-side of small aliphatic amino acids (gly, ala, ser)
20
Q
What are the exopeptidases
A
CPA - cleaves neutral amino acids from the carboxyl end.
CPB - cleaves basic amino acids from the carboxyl end
21
Q
Brush border hydrolysis of oligopeptides
A
The oligopeptides produced by
hydrolysis in the lumen are further hydrolyzed by a family of oligopeptidases that
are localized in the brush border membrane. These enzymes are all glycoproteins
and the carbohydrate moiety ensures that the peptidase is oriented so that the
active site is accessible. The final products of hydrolysis by the brush border
peptidases are a mixture of free amino acids, dipeptides, and tripeptides.
22
Q
Describe transport of amino acids
A
A. Luminal Membrane. Several specific transport proteins exist in the brush border
membrane for transporting amino acids into the intestinal cell. (Note: Many of these
transporters are also expressed in the brush border membranes of renal tubules.) These
systems co-transport Na
+
(provides driving force for uptake, similar to intestinal glucose
uptake) and include the following transporter classes:
1. Neutral Amino Acid Transporter
2. Aromatic/Hydrophobic Amino Acid Transporter
3. Imino Acid Transporter
4. Acidic Amino Acid Transporter
5. Basic Amino Acid Transporter
B. Basolateral Membrane. There is a different set of transport proteins in the
basolateral membrane. Most of these are Na
+
independent.
23
Q
Describe abnormalities in protein digestion and absorption
A
A. Disorders of Digestion (usually pancreatic or intestinal in origin)
1. Primary: Parietal cell deficiency
Secondary: Long-term use of proton pump inhibitors (PPIs)*
2. Zollinger-Ellison Syndrome (gastrin-secreting tumors in pancreas)
3. Pancreatic Insufficiency (cystic fibrosis; pancreatitis)
4. Congenital enterokinase deficiency
5. Trypsinogen-trypsin deficiency
B. Disorders of Absorption
1. Hartnup’s Disease (Neutral Amino Acid Transporter)
2. Cystinuria (Basic Amino Acid Transporter). This is the most common
disorder in amino acid metabolism. It is characterized by the loss of cystine,
ornithine, arginine, and lysine in the urine. The low solubility of cystine at acidic
pH values also results in kidney stones.
3. Prolinuria (Imino Acid Transporter)
4. Acquired disorders (Crohn’s disease, celiac spru, radiation enteritis,
infection)
24
Q
Describe the effects of longterm PPI use
A
*NOTE: Because the parietal cells targeted by PPIs also produce the Intrinsic Factor required for
absorption of dietary Vitamin B12, long-term PPI usage can result in B12 deficiency, which can
result in anemia and peripheral sensory and motor neural deficiencies. This can easily be
treated by B12 injections.
25
OVERVIEW OF GENERAL PATHWAY for catabolism of amino acids
1. removal of the α-amino group.
2. nitrogen can be incorporated into other compounds or excreted.
The carbon skeletons that result from the removal of the amino group are major
metabolic intermediates of carbohydrate metabolism and the citric acid cycle.
26
Types of Reactions for Removal of α -Amino Groups
1. Deamination Reactions produce free ammonia
2. Transamination Reactions transfer amino groups to a common
acceptor, usually α -ketoglutarate
27
Fate of Carbon Skeletons in amino acid catabolism
form major metabolic intermediates that can be converted into glucose or ketones. The
carbon skeletons of the 20 common amino acids funnel into seven metabolites.
Some of the amino acids (e.g., leucine, tryptophan, isoleucine, phenylalanine) are
fragmented in such a way that they produce more than one of these seven metabolites.
```
a. Pyruvate is produced by the degradation of alanine, serine,
glycine, cysteine, and threonine.
b. Oxaloacetate is produced by the catabolism of aspartate and
asparagine.
Alanine
Serine Cysteine
Threonine
Pyruvate
(3 C)
Asparagine Aspa
rtate Oxaloacetate
```
28
What is the fate of glucogenic amino acids?
Glucogenic Amino Acids have carbon skeletons that are converted to either pyruvate, oxaloacetate, α -ketoglutarate, succinyl-CoA or fumarate. The most important of these amino acids are alanine,
aspartate, and glutamate.
29
What amino acids produce pyruvate?
alanine, serine, glycine, cystein, and threonine
30
What amino acids produce oxaloacetate?
catabolism of aspartate and asparagine
| asparagine --> aspartate --> OAA
31
What amino acids produce alpha-ketoglutarate?
glutamine, proline, arginine and histidine converted to glutamate and then transaminated to alpha-ketoglutarate
32
What amino acids produce succinyl coA?
degradation by methionine, isoleucine, and valine, which form propionyl CoA then methyl malonyl CoA and finally succinyl CoA
33
What amino acids produce fumerate?
phenylalanine forms tyrosine which forms homogentisate to fumarylacetoacetate which breaks down to acetoacetate and fumerat
34
What are ketogenic amino acids
amino acids that are degrade to acetyl CoA or acetoacetyl CoA. These products can be used for the synthesis of ketones (and fatty acids)
Leucine and lysine are purely ketogenic
isoleucine, phenylalanine, tyrosine and tryptophane are both ketogenic and glucogenic
35
Deamination
These reactions generate free ammonia which is toxic
and must be "fixed" or detoxified. There are two general classes of deamination
reactions: nonoxidative and oxidative.
36
Non-oxidative deamination
Dehydration of Serine and Threonine eliminates ammonia in a
reaction involving the side chain hydroxyl group and the hydrogen
attached to the α -carbon atom (these enzymes require pyridoxalP).
Serine → pyruvate + NH4+ + H2O Threonine → α -ketobutyrate + NH4+
+ H2O
b. Hydrolytic deamination of side chain amide groups on Asparagine
(via asparaginase) and Glutamine (via glutaminase). (These enzymes do
not require pyridoxal phosphate)
Asparagine + H2O → Aspartic Acid + NH4+
Glutamine + H2O → Glutamic Acid + NH4+
c. Direct deamination of Histidine and Glycine
(no requirement for pyridoxal phosphate)
Histidine → Urocanate + NH4+
Glycine → CO2 + NH4+
37
Oxidative Deamination
Oxidative Deamination of glutamic acid to produce α -ketoglutarate and
ammonia. This reaction requires NAD+
or NADP+ as a coenzyme. The enzyme glutamate dehydrogenase is present in the mitochondrial matrix in very high concentrations. glutamate + NAD+
→ α -ketoglutarate + NADH + H+ + NH4+
38
Transamination Reaction
These reactions do not directly release free ammonia, but rather transfer the α -amino group from amino acids to some α -keto acid acceptor.
Transaminases require pyridoxal-P as a coenzyme. This is the major pathway
for removal of nitrogen from amino acids. There are transaminases for most of
the amino acids. With the exception of the branched chain amino acids (val, ile,
leu), the catabolism of most dietary amino acids starts in the liver. There is no
branched chain amino acid transaminase in the liver. The concentration of
branched chain amino acids in the blood leaving the liver is as high as it was in
the portal blood bringing dietary amino acids to the liver.
39
Strategy of Transamination Reactions
Strategy of Transamination Reactions is to transfer the amino nitrogen from a diverse group of donor amino acids to a smaller number of -keto acid acceptors so that there can be a central pathway for disposal. Most transaminases use α -ketoglutarate as the amino acceptor. Transaminases are usually named by the amino acid which is the amino donor. These reactions are reversible, so the direction of the reaction can be altered by changes in concentrations of substrates and products.
40
Substrate Specificity of Transaminases
Each transaminase is specific for
one or a few amino acid nitrogen donors. Although the nitrogen acceptor for
most transaminases is α -ketoglutarate, oxaloacetate and pyruvate are also two
very important amino group acceptors. Quantitatively, the two most important
transaminases are AST and ALT
41
ALT
Alanine transaminase (ALT) also known as
glutamate:pyruvate transaminase (GPT, or SGPT when referring to
its presence in the serum). Note that these reactions are reversible.
In skeletal muscle pyruvate is the major acceptor for amino groups
from glutamate, thus producing large quantities of alanine (which
is transported to the liver). In the liver, alanine donates this amino
group back to α -KG to form glutamate.
42
AST
Aspartate transaminase (AST) also known as
glutamate:oxaloacetate transaminase (GOT or SGOT). This
reaction is especially important in the liver where oxaloacetate acts
as an acceptor for some of the amino groups that have been
funneled into glutamate. The product of the reaction in liver is
aspartate, which is a nitrogen donor for urea synthesis.
43
ROLE OF PYRIDOXAL PHOSPHATE IN AMINO ACID METABOLISM
A. Vitamin B6 is the Precursor for Pyridoxal Phosphate - Vit B6 exists in a
number of forms, including pyridoxine and pyridoxal. The coenzyme forms of B6
have phosphate esterified to the methoxy side chain.
B. Function. Pyridoxal phosphate acts as a coenzyme for many enzymes that
catalyze reactions involving transformations around the α-carbon atom of amino acids. For example transaminases, decarboxylases, deaminases, racemases and aldolases (dehydratases).
C. Mechanism. Pyridoxal phosphate activates the α-carbon atom and makes
one of the three bonds attached to the α -carbon atom labile.
D. Role of Pyridoxal-P in Transaminase Reactions. It acts as a carrier of
amino groups in a two-step reaction. Pyridoxal-P first accepts the amino group
from a donor amino acid to form pyridoxamine-P. In the second half of the
reaction, the amino group is donated to an α-keto acid acceptor (usually α -
ketoglutarate), thus regenerating pyridoxal-P and forming a new amino acid
(usually glutamate).
44
Drug-Induced Vitamin B6 Deficiency
Isoniazid is a drug that is used for the treatment of tuberculosis. It reacts
with pyridoxal and thus makes it unavailable for phosphorylation by
pyridoxal kinase. Transaminase activities may be lower than normal in
patients receiving isoniazid.
Isoniazid + Pyridoxal → Inactive compound
2. Penicillamine is used to treat Wilson's disease, a Cu
+2
storage disease.
Penicillamine inactivates pyridoxal and thus can affect transaminase
activity. Such patients require extra pyridoxine to normalize transaminase
levels.
Penicillamine + Pyridoxal → Inactive compound
45
Conditions Associated with Increased or Decreased Serum Levels of
Transaminases
1. Increased serum levels: myocardial infarct (AST); hepatitis (AST, ALT); alcoholic liver damage (AST, ALT); liver cancer (AST, ALT); muscular dystrophy (ALT)
2. Decreased serum levels: nutritional pyridoxine deficiency; patients on hemodialysis.
46
Role of Muscle in amino acid metabolism
Muscle is the major tissue supplying amino acids to the blood. Alanine
and glutamine collectively account for more than 50% of the amino
acids released by muscle. Muscle release of alanine and glutamine is
directly linked to the metabolism of branched chain amino acids. The
release of alanine and glutamine increases significantly during starvation.
blood, making the kidney a gluconeogenic
organ.
47
The role of liver in amino acid metabolism
Liver is most efficient in taking up alanine released by muscle and
intestine. The liver ultimately incorporates the amino group of alanine into
urea and uses the carbon skeleton for gluconeogenesis. Liver also takes
up small amounts of glutamine, especially in the postabsorptive state.
Ammonia is transported to the liver for urea synthesis in the form of
either glutamine (from most tissues) or alanine (from muscle).
48
The role of small intestine in amino acid metabolism
Small intestine takes up large quantities of glutamine that come from
dietary protein or from skeletal muscle. Glutamine is the major fuel for
the intestine. Two of the products of glutamine metabolism in the
intestine are alanine and citrulline, which are released into the circulation.
49
The role of the kidneys in amino acid metabolism
Kidney takes up glutamine that is released by muscle and uses it for
ammoniagenesis. The release of ammonia and subsequent titration with
protons generates ammonium ions that play a role in acid-base balance
and help conserve cations (discussed in later section on nitrogen
50
BRANCHED CHAIN AMINO ACID (BCAA) METABOLISM.- Transamination and Decarboxylation
The branched chain amino acids (Valine,
Isoleucine and Leucine) are not extracted by the liver because there is little or no BCAA
transaminase (BCAT) in the liver. They leave the liver and enter the systemic
circulation. Transamination and decarboxylation both occur in skeletal muscle. Except
for the cytosolic isozyme of BCAT, all enzymes involved in BCAA catabolism are
mitochondrial. BCAAs are essential dietary amino acids.
51
Describe the usage of BCKAs as fuel
BCKAs are used as Fuel by Muscle, Kidney, Liver and Brain. Whereas transamination can only occur in skeletal muscle, the resulting BCKA (branched chain keto acid) can be further metabolized in skeletal muscle or they can leave the muscle and be taken up by liver, kidney or brain and be used as fuel by these tissues.
52
Degradation of BCKA
Degradation starts with transamination and is followed by oxidative decarboxylation of the respective BCKA. There are two BCAT isozymes: BCATm (mitochondrial) and BCATc (cytosolic). BCATm is expressed in most tissues (very little in liver), whereas BCATc is primarily restricted to the CNS. The majority of BCAT activity is in skeletal muscle.
53
The Branched Chain α-Ketoacid Dehydrogenase Complex
The Branched Chain α-Ketoacid Dehydrogenase Complex (BCKA-DH) is similar in structure and mechanism to the pyruvate dehydrogenase complex and the α-KG dehydrogenase complex. All are localized in the mitochondria, consist of three enzyme activities and require five coenzymes for catalytic activity. The E1 subunit, BCKA decarboxylase, requires TPP as a prosthetic group. The E2 subunit, dihydrolipoyl transacetylase, uses lipoic acid as a prosthetic group. The E3 subunit, dihydrolipoyl dehydrogenase, has FAD as a prosthetic group. In addition to the prosthetic groups, NAD+ and CoASH are coenzymes for this enzyme complex. The E3 subunit is the identical gene product for BCKA DH, pyruvate DH and α -KG DH. Similar to pyruvate DH, the BCKA dehydrogenase complex also has a specific kinase and phosphatase associated with the complex which phosphorylates and dephosphorylates the E1 subunit. The BCKA-DH complex catalyzes the rate-limiting step in BCAA catabolism and its activity is highly regulated (discussed below).
54
Assimilation of Propionyl~CoA into the Citric Acid Cycle
(1) Propionyl-CoA carboxylase (requires biotin)
(2) Epimerase
(3) Methylmalonyl-CoA mutase (requires vitamin B12
55
Interorgan Relationships in BCAA Metabolism
Catabolism of BCAA requires several organs. Liver has a low capacity for
transamination of BCAA, but has a high capacity for the oxidation of BCKA. On
the other hand, skeletal muscle has a high capacity for transamination of BCAA
and a low capacity for the catabolism of BCKA. Consequently, much of BCAA
transamination occurs in skeletal muscle, where the amino nitrogens end up on
alanine and glutamine and the BCKA are released into blood where they are
taken up by the liver, heart, kidney or brain for further catabolism. Major uses of
BCAA are Heart: energy; Brain, energy and lipid synthesis; Kidney, energy\
56
Regulation
Regulation of BCAA Metabolism is exerted at the level of BCKA-DH by 2 mechanisms:
1. The ratio of NADH/NAD+ and acyl~CoA/CoA control the flux of BCKA through the BCKA-DH step (feedback or product inhibition). BCKA + NAD+
+ CoASH → Acyl~CoA + NADH + CO2
2. Phosphorylation-Dephosphorylation also regulates the activity. The enzyme is
active in the dephospho-form and inactive in the phospho-form. Only one of the
three enzymes (E1) is phosphorylated. Alteration of the phosphorylation state permits rapid modulation of enzyme activity in response to changes in a number of dietary and hormonal factors.
The BCKA-DH Kinase is very sensitive to the concentrations of the keto acid of leucine.
High concentrations of BCKA inhibit the kinase. This inhibition is of physiological
significance. For example, after a high protein meal, the concentrations of BCKAs
increase and inhibit the kinase. This inhibition prevents phosphorylation and ensures that the BCKA DH will remain in its active form. Conversely, when the diet is free of protein, BCAA must be conserved since these are essential amino acids. Conservation, rather than degradation, is accomplished by phosphorylation and inactivation of BCKA-DH.
57
Physiological Significance of BCAA Metabolism
Catabolism of BCAA is of considerable importance. Transamination in skeletal muscle provides amino nitrogen for the synthesis of alanine and glutamine, which are released in large quantities from muscle. Alanine and glutamine are key substrates for gluconeogenesis and
ammoniagenesis in the liver and kidney, respectively. Additionally, glutamine is the
major fuel for the intestine. Oxidation of BCAA in brain provides carbon fragments for lipogenesis, which is particularly important in the synthesis of myelin. In cardiac tissue, BCAA are completely oxidized to CO2 and H2O.
58
Disorders of BCAA Metabolism
1. Propionic Acidemia - deficiency of propionyl~CoA carboxylase
2. Methylmalonic Acidemia - deficiency of methylmalonyl-CoA mutase.
Characteristics of these disorders:
a. High levels of propionic and methylmalonic acid in blood and urine
b. Metabolic acidosis
c. Urinary excretion of acyl-carnitines
d. Secondary effects: Hypoglycemia, Hyperammonemia
59
Treatment of Propionic and Methylmalonyl Acidemia:
a. Restricted protein intake
b. Prevention of a catabolic state (e.g., infection)
c. Add Biotin (a cofactor for propionyl-CoA carboxylase)
d. Add Vitamin B12 (cofactor for methylmalonyl-CoA mutase)
e. Add Carnitine (to free up CoASH and allow β-oxidation)
60
Maple Syrup Urine Disease (MSUD)
Deficiency in BCKA-DH complex
Symptoms: Urine has the odor of maple syrup or burnt sugar; elevated plasma and urine levels of leu, ile, val and their corresponding α - ketoacids; branched chain α -hydroxy acids in urine; vomiting; lethargy; brain damage; death by end of first year. Autopsy shows brain edema, lack
of myelin and a reduction in total lipids.
Genetics: Autosomal recessive, affects both males and females; carriers are asymptomatic.
Diagnosis: circumstantial evidence cited under symptoms; assay BCKA-DH activity of isolated leukocytes or fibroblasts. Usually no abnormalities in blood or urine at birth because it has been regulated by
the maternal circulation but may show up about a week later.
61
Treatment of MSUD
Treatment: Use a diet with no BCAA until blood levels have fallen to
normal and then supplement to maintain near normal concentrations.
Avoid conditions that increase breakdown of tissue protein (e.g.,
infection). It is difficult to do the necessary analytical work and prepare
proper diets for treatment.
62
Biochemical significance of MSUD
A defect may exist in any one of the multiple
reactions catalyzed by the BCKA-DH complex. (However, a defect in the
E3 subunit would also affect the PDH and α KG-DH complexes.) The
high concentration of BCAA may have effects on the transport of other
amino acids and distort the intracellular amino acid pools. There seems to
be a secondary effect on the synthesis of proteolipids, cerebrosides and
myelin, although the basis for these effects is unknown.
63
Other Defects in BCAA Metabolism
a. Hypervalinemia - inability to transaminate valine; no
apparent defect in the ability to transaminate leu and ile.
b. Intermittent branched chain ketonuria - an intermittent
defect in BCKA-DH affects all three of the amino acids. Probably
a variant of Maple Syrup Urine Disease.
64
The role of folic acid and S-adenosyl methionine in amino acid metabolism
1. Several steps in metabolism require the transfer of a one-carbon unit from one compound to another. These reactions are especially important in amino acid metabolism and in purine and pyrimidine
metabolism.
2. Single carbon atoms exist in a number of oxidation states.
3. The single carbon units can be transferred from carrier compounds
such as folic acid and S-adenosyl methionine (SAM) to specific acceptors that are being modified. This group of carriers is known as the "one-carbon pool".
4. Formaldehyde (H2CO) is toxic; formic acid (HCOOH) is nonreactive. Thus, a major function of the one-carbon pool is to maintain formaldehyde and formic acid in a non-toxic but active state which can be
used by specific enzymes.
65
structure of folic acid
Folic acid contains a bicyclic, nitrogenous moiety
(pteridin), p-aminobenzoic acid (PABA), and one or more glutamate
residues.
66
What is THF
The active form of folic acid, tetrahydrofolate (THF) is produced by the action of dihydrofolate reductase in a two-step reaction requiring NADPH.
The carbon units carried by THF are bound to the N5 or N10 or both of these nitrogen atoms.
The source of carbon units are the amino acids tryptophan, histidine, glycine and serine.
67
Synthesis of SAM
THF can carry methyl groups but its transfer potential is not sufficiently high for
most biosynthetic reactions. The preferred "activated methyl" carrier in most
methylation reactions is SAM.
1. Synthesis of SAM - The methyl group originates in the side chain of methionine. In order to "activate" the methyl group, the sulfur atom of themethionine side chain is attached to C-5 of ribose in the adenosine
molecule.
68
synthesis of creatine
This represents the body’s greatest use of SAM. The
biosynthetic pathway involves two enzymes that are located in different organs:
arginine-glycine amidinotransferase (AGAT) in kidney and guandinoacetate
methyltransferase (GAMT) located in liver. Creatine in various cell types,
including muscle and brain, is phosphorylated by creatine kinase (CK; also known
as creatine phosphokinase, or CPK).
69
Synthesis of carnitine
Carnitine is somewhat unusual in that it is
derived from methylated lysine residues in proteins rather by modification
of free lysine. SAM is used to add methyl groups to the lysine side chain
within proteins, followed by proteolysis to liberate trimethyllysine, which
undergoes additional reactions to form carnitine.
70
How are the nonessential amino acids made?
Nine of the 11 non-essential amino acids are synthesized from
intermediates in glycolysis or the citric acid cycle. Transamination and
amidation reactions are important in these pathways. The remaining two, tyrosine
and cysteine, are formed from the essential amino acids phenylalanine and
methionine, respectively.
71
synthesis of alanine
Alanine, is synthesized by transamination
of an amino group to the α-keto acids pyruvate. Transaminase-catalyzed reactions are readily
reversible.
72
synthesis of aspartate
is synthesized by transamination
of an amino group to the α-keto acids oxaloacetate. Transaminase-catalyzed reactions are readily
reversible.
73
synthesis of glutamate
is synthesized by transamination
of an amino group to the α-keto acids α -
ketoglutarate, respectively. Transaminase-catalyzed reactions are readily
reversible. It can also be synthesized by the reverse of the oxidative deamination reaction catalyzed by glutamate dehydrogenase.
74
synthesis of proline
from glutamate
75
synthesis of glutamine
from glutamate and ammonia
enzyme: glutamine synthetase
requires 1 ATP
76
synthesis of arginine
from glutamate
77
synthesis of asparagine
from aspartate and glutamine
enzyme: asparagine synthetase
requires one ATP
78
synthesis of serine
from 3 phosophoglycerate
| serine and glycine are interconvertible through the enzyme: serine hydroxylmethyltransferase
79
synthesis of glycine
serine and glycine are interconvertible through the enzyme: serine hydroxylmethyltransferase
80
synthesis of cystein
from methionine, involving SAM, Vitamin B12 and folic acid. can be made from homocystein which is created from demethylation of SAM and removal of adenosine
81
Synthesis of methionine
homocystein can be used to regenerate methionine in a remethylation reaction requiring vit b12 and folate.
Methionine synthetase requires B12
methionine can also be regenerated by a remethylation reaction in which the methyl donor is betaine, which is derived from choline
82
functional folate deficiency
deficiency of methionine synthase or a vitamin b 12 deficiency can cause this because it is the only reaction in which 5methyl THF can be converted to THF. In the absece of this reaction, folate accummulates as 5-methylTHF which cannot be used in any other reaction that requires folate
83
Synthesis of tyrosine
from phenylalanine
enzyme: phenylalanine hydroxylase
a. Monooxygenase - requires molecular oxygen; one atom is reduced to a hydroxyl group and incorporated into the ring; the other oxygen atom is reduced to water.
b. Requires reduced tetrahydrobiopterin (THB or BH4) as a source of reducing power for molecular oxygen. In each cycle of the reaction tetrahydrobiopterin becomes oxidized to dihydrobiopterin (DHB).
c. NADPH is required for regeneration of reduced biopterin. This reaction is catalyzed by dihydrobiopterin reductase.
84
Phenylketonuria
a. Classical PKU - deficiency in phenylalanine hydroxylase
b. Atypical PKU - deficiency in biopterin or in dihydrobiopterin
reductase
c. Maternal PKU - damage to fetus by maternal phenylketones
85
synthesis of arginine
it requires the small intestines and kidney
Arginine is not an essential amino acid except in infancy or in cases where intestinal or renal function is seriously impaired (e.g., injury, infection, or surgical resection). It is synthesized by the liver as an intermediate in the urea cycle. However, the activity of arginase is so high that arginine is cleaved to urea and ornithine as rapidly as it is formed, and thus the urea cycle does not serve as a pathway for the net biosynthesis of arginine. The first two enzymes of the urea cycle are found in the intestinal cells, where the first two reactions of arginine biosynthesis occur, resulting in the production of citrulline. The ornithine required for biosynthesis of citrulline is derived primarily from the intestinal
catabolism of glutamine. Citrulline is released from the enterocyte into the circulation, and its concentration in plasma is an indicator of functional small intestinal mass. It is extracted by the kidney which contains the next two enzymes of the urea cycle in the proximal tubules. Thus, in the kidney, citrulline is efficiently converted to arginine, which is released into the blood.
86
Ammonium ion metabolism
A. Source. The most common source of ammonium ions (NH4
+
) is from
deamination of amino acids, which occurs in all tissues. Metabolism by colonic
bacteria also is a significant source of NH4
+
that enters the portal blood.
B. Toxicity. Ammonia is extremely toxic to the CNS, and various
mechanisms exist for its detoxification. In brain, the synthesis of glutamine is
used to convert ammonia into a nontoxic organic form which can be used as a
source of amino groups for biosynthetic purposes. Excess glutamine can be
degraded in liver to α-KG and NH4
+
where the NH4
+
is converted to urea.
C. Detoxification of Ammonium Ions in Extrahepatic Tissues by Glutamine
Synthetase.
1. Reaction catalyzed by glutamine synthetase (GS):
87
Tissue Distribution and Subcellular Localization of GS
present in cytosol of all tissues, but especially enriched in brain (where
ammonia is extremely toxic) and muscle (where turnover of muscle
protein produces substantial amounts of NH4+).
88
Functions of flutamine
a. In all tissues, glutamine is a precursor form of nitrogen for
various pathways, especially for purine and pyrimidine biosynthesis.
b. It serves as a nontoxic transporter of ammonium ion
from extrahepatic tissues to liver where it can be converted to urea.
c. In gut, it serves as the major fuel for the enterocyte and
also is partially metabolized to provide precursors for other amino acids.
In the immune system, it also is a major fuel for macrophages and
lymphocytes.
d. In kidney, it is important in maintaining acid-base
balance. The hydrolysis of the amide group in the side chain of glutamine
by glutaminase provides a sink for protons. This is particularly important
in conditions of metabolic acidosis.
89
ROLE OF THE LIVER IN DETOXIFICATION OF AMMONIA
Efficiency of Liver in Removing Ammonium Ions - In the post absorptive state,
the concentration of ammonium ion in the portal blood is approximately 0.3mM.
As the blood moves through the liver, this concentration can be increased to as
high as 1.0mM. However, by the time blood leaves the liver and enters the
systemic circulation, the concentration has been reduced to approximately
0.02mM.
90
Periportal hepatocytes
1. Periportal hepatocytes are near the portal vein and receive a
blood supply that is very rich in nutrients. These hepatocytes are
enriched in glutaminase and the enzymes of urea synthesis.
Glutaminase is allosterically activated by ammonium ions, so that the
ammonium ion entering the hepatocytes also activates the enzyme which
converts glutamine to ammonium ion and glutamate. The bulk of the
ammonium ion is removed from the circulation by the periportal
hepatocytes. The Km of carbamoyl-P synthetase I for NH4
+
is
approximately 1.0 mM.
91
Perivenous hepatocytes
Perivenous hepatocytes make up one to three layers of cells that
surround the central vein. These cells are enriched in glutamine
synthetase, which has a low Km for ammonium ion. Therefore, the
ammonium ions that do not get converted to urea in the periportal cells
get trapped as glutamine in the perivenous cells.
92
THE UREA CYCLE
The Major Mechanism for Ammonium Ion Detoxification - The liver is the only
organ that contains all of the enzymes of the urea cycle in significant amounts,
and consequently is the site of virtually all urea synthesis. Both mitochondrial
and cytosolic enzymes are required
93
Urea Cycle Enzymes
CPS-I: Carbamyl phosphate synthetase-I
OTC: Ornithine transcarbamylase
ASS: Argininosuccinate synthetase
ASL: Argininosuccinate lyase
ORNT1: Mitochondrial ornithine/citrulline antiporter
Citrin: Mitochondrial aspartate/glutamate antiporter
NAGS: N–Acetyl glutamate synthetase
94
Synethesis of carbamoyl phosphate
occurs in the mitochondria). Carbamoyl
phosphate is synthesized in a complex reaction that is catalyzed by carbamoyl
phosphate synthetase I. This enzyme requires N-acetylglutamate (NAG), an
allosteric activator, for activity. The consumption of two molecules of ATP
makes the synthesis of carbamoyl-P irreversible. A part of the energy in ATP is
conserved in the high energy carbamoyl~P bond (a mixed anhydride)
The carbamoyl phosphate synthetase is referred to as CPS-I to distinguish this
enzyme from CPS-II, an isozyme that is found in the cytosol and participates in
pyrimidine synthesis.
95
Formation of citrulline
1. Both ornithine and citrulline are basic amino acids that participate in the
urea cycle. Ornithine is regenerated with each turn of the cycle.
2. The reaction is driven by the release of high energy phosphate from
carbamoyl~P.
3. The product, citrulline, is transported into the cytosol where the remainder
of the reactions occur.
96
Formation of Arginosuccinate
The condensation of aspartate with citrulline is catalyzed by argininosuccinate
synthetase:
Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi
PPi → 2 Pi
1. The α-amino group of aspartate provides the second nitrogen atom in urea.
2. The formation of argininosuccinate is driven by the cleavage of ATP to AMP and PPi
. Since ubiquitous pyrophosphatases hydrolyze the P~ Pi to 2Pi
, this reaction is shifted in the direction of argininosuccinate synthesis
by the hydrolysis of two high energy phosphate bonds.
Occurs in cytosol
97
Cleavage of argininosuc
Cleavage is catalyzed by argininosuccinate lyase.
1. The arginine formed from argininosuccinate is the immediate precursor of
urea. The fumarate produced in this reaction provides a link with other
pathways.
Argininosuccinate → Arginine + Fumarate
occurs in cytosol
98
Cleavage of arginine
Arginine → Ornithine + Urea
1. The enzyme arginase (type I isozyme) is required for the cyclic nature of
the pathway. It is present primarily in liver but can be induced by
cytokines and other stimuli in many other tissues.
2. Urea diffuses from the liver and is transported in the blood to the kidneys
where it is filtered and excreted. About 25% diffuses into the colon where
it is a substrate for microbial urease.
3. Ornithine is transported back into the mitochondria where it can begin to
participate in another cycle of urea synthesis.
99
Stoichiometry of Urea Synthesis
Aspartate + NH3 + CO2 + 3ATP → 3H2O + urea + fumarate + 2ADP + AMP + 2Pi
+ PPi
1. The equivalent of 4 high energy phosphate bonds is consumed for every
molecule of urea synthesized.
2. One N is supplied by ammonium ion and the other by aspartate.
Aspartate carries the amino group contributed by other amino acids (e.g.,
alanine) via transamination.
100
Regulation of Urea Cycle
1. Short term regulation of urea synthesis is exerted at the level of
carbamoyl phosphate synthetase I (CPS-I). This enzyme is allosterically
regulated by N-acetylglutamate (NAG). NAG is synthesized inside the
mitochondria from glutamate and acetyl-CoA. The reaction is catalyzed
by NAG synthase, which, in turn, is activated by arginine.
Acetyl~CoA + Glutamate → N-acetylglutamate + CoA
2. Long-term regulation of urea synthesis depends on changes in the levels
of urea cycle enzymes, primarily via changes in transcription of the
corresponding genes. Enzyme levels change in response to changing
levels of dietary protein. E.g., an increase in the protein content of the diet
over several days, or increased protein catabolism, increases the activity
and amount of all five enzymes of the urea cycle.
(look at table)
101
Deficiency of enzymes in the urea cycle
Collectively the urea cycle deficiencies occur at a
frequency of 1 per 25,000 live births, and they usually become manifested during
the neonatal period. Inherited defects of each of the enzymatic steps involved in
urea synthesis have been described. All of the defects (with the exception of
arginase deficiency) results in severe hyperammonemia. High levels of ammonia
are extremely toxic to the CNS, If hyperammonemia is not readily treated, the
infant develops severe mental retardation
102
Disorders of the urea cycle
1. Hyperammonemia Type I (defect in CPS-I)
2. Hyperammonemia Type II (defect in OTCase)
3. Citrullinemia (defect in AS)
4. Argininosuccinic aciduria (defect in AL)
5. Hyperargininemia (defect in Arginase)
103
Treatment of urea cycle disorders
1. Promote alternative ways of N-elimination by administration of:
a. Sodium benzoate
b. Sodium phenylacetate
2. Restrict dietary protein intake, and supplement with essential amino acids.
3. Prevent catabolic situations (e.g., infections)
4. For deficiencies in certain enzymes, provide arginine (to prime the urea
cycle and to promote the formation and excretion of certain urea cycle
intermediates)
5. Liver transplantation
6. Gene therapy in future
104
Alternative pathways of nitrogen excretion
Administration of
phenylacetate allows amino acid nitrogen in the form of glutamine to be excreted
as phenylacetylglutamine. Administration of benzoate allows amino acid
nitrogen in the form of glycine to be excreted as hippuric acid. Because the
nitrogen atoms in glycine and glutamine are in equilibrium with nitrogen atoms in the free amino acid pool, these amino acids can act as “sinks” for metabolically
generated waste nitrogen (NH4+).
105
Causes of hyperammonemia
Inherited defects in the urea cycle represent a relatively minor cause of
hyperammonemia. Other causes include:
• Liver Disease
• Organic Acidemias—Due to accumulation of Co-A derivatives of organic acids
• Congenital Lactic Acidosis--characterized by increased lactate concentrations
• Fatty Acid Oxidation Defects
• Dibasic Amino Acid Transport Defects--mitochondrial ornithine transporter
defect
• Reye’s Syndrome--acquired disorder associated with viral infection, exacerbated
by aspirin in children (now relatively rare since precautions against giving aspirin
to children were adopted)
• Some Drugs--Valproate, Chemotherapy, Salicylate
106
Renal ammonia synthesis
Glutamine serves as the precursor for most of the urinary ammonium ion. Ammonium is produced in kidney through two successive reactions that first remove the amide group of the side chain of glutamine and then the α-amino group of glutamate. These reactions are catalyzed by glutaminase and glutamate dehydrogenase, respectively. The carbon skeleton, α-ketoglutarate, can be utilized for renal gluconeogenesis via the TCA Cycle and renal PEPCK
107
Ammoniagenesis in Kidney
1. The kidney excretes acid that is normally generated in metabolism. However, secretion of protons ceases when the proton gradient exceeds about 3 pH units. If we simply excreted protons via an unbuffered urinary filtrate, we would require a urine volume of about 1000 liters/day.
2. Excretion of protons via a buffered solution greatly reduces urine volume. Principal buffers are phosphate ion (HPO4
-2/H2PO4-) and ammonium ion (NH3/ NH4+). Respective counterions are Na+ and Cl-
3. Normally, excreted protons are buffered by HPO4-2 and excreted as H2PO4- plus Na+
During acidosis, however, this initially results in increased loss of Na+, followed by loss of K+.
4. To prevent loss of critical cations, renal ammoniagenesis increases. Thus, protons will be buffered by NH3 and excreted as NH4+ plus Cl-
108
Creatinine formation
A. Formed in a Non-enzymatic Reaction via spontaneous cyclization of either
creatine or creatine phosphate.
109
Creatinine excretion
Amount of creatinine excreted is proportional to skeletal muscle mass.
When muscle mass decreases from paralysis, atrophy, dystrophy, etc., the
creatinine content of the urine also decreases.
C. Creatinine Levels are Used to Assess:
1. Kidney Function - Creatinine is normally removed from the blood
and excreted rapidly. Therefore, any rise in creatinine levels in the plasma is an
indicator of kidney malfunction.
2. Accuracy of 24-Hour Urine Specimens - Because creatinine
excretion over a 24-h period is so constant for each individual with normal renal
function, it is often used to assess the reliability of a urine collection, where too
little creatinine may indicate an incomplete sample.
110
Uric Acid formation
Humans have no enzymes that can open up the purine ring system and degrade it into
smaller fragments for excretion. The strategy used by the body for degrading purines is
to remove substituents from the purine ring system and to make the ring as soluble as
possible by oxidation of carbon atoms in the ring. The nucleotide is first converted to a
nucleoside and then to a purine base.
111
Gout
Gout: a condition that results from formation of uric acid crystals that elicit painful
inflammation in feet and hands.
1. Enzyme Defects Resulting in Overproduction of Uric Acid:
a. Partial deficiency in HGPRT (also results in Lesch-Nyhan Syndrome*)
b. Abnormally High Activity of PRPP Synthetase (involved in purine synthesis)
c. Glucose-6-phosphatase Deficiency (causes are complex but involve excess
production of purines and impaired uric acid excretion)
112
Lesh-Nyhan Syndrome
characterized by elevated uric acid, uncontrolled muscular
movements and a variety of other CNS defects, and compulsive self-mutilation. As the gene for
HGPRT is on the X-chromosome, this disease is essentially restricted to males.
113
Treatment of Cout
1. Treatment with Allopurinol
a. Structure: Allopurinol is an analog of hypoxanthine.
b. Mechanism: It acts as a suicide inhibitor of xanthine oxidase by conversion to
alloxanthine and tight binding to the active site.
Allopurinal inhibits formation of xanthine from hyposanthine and the formation of uric acid from xanthine
Note: A new xanthine oxidase inhibitor [Uloric (febuxostat); not a purine analog] was approved
by the FDA in 2009. This was the first new drug approved for treatment of gout in 45 years.
2. Treatment with Probenecid --- drug that facilitates urinary excretion of uric acid.
