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Proteins
Nitrogen balance
Seminar No. 4
- Chapter 10 -
1
Overall metabolism of proteins
• Schemes on p. 53 and 54
• all proteins in the body are continuously degraded (metabolized)
and newly synthesized
• free AA from food, tissue proteins and non-essential AA from
synthesis make AA pool
• AA pool is used for:
new body proteins
specialized products (amines, NO, porphyrines, NA bases ...)
catabolic proceses (energy gain)
2
AA pool
~ 80 % in muscles
~ 10 % in liver
~ 5 % in kidney
~ 5 % in blood
AA pool is not reserve !!
There is not a specific protein reserve in human body in contrast
to saccharides (liver glycogen) and lipids (adip. tissue) !!
3
AA in blood
Resorption phase
• predominate Val, Leu, Ile
• liver does not take them up from circulation (no specific
aminotransferases in liver for Val, Leu, Ile)
Postresorption phase and fasting
• predominate Gln and Ala
• released from muscles (Gln + Ala) and liver (Gln)
4
There are eight essential aminoacids
• valine
• phenylalanine
• leucine
• tryptophan
• isoleucine
• lysine
• threonine
• methionine
Conditionally essential aminoacids
Biological value of proteins (BV) the
histidine, arginine (in childhood and youth)
amount of endogenous proteins made in
alanine, glutamine (in metabolic stress)
body from 100 g of dietary proteins
about 30 % of methionine requirement can be made up by cysteine
about 50 % of phenylalanine requirement can be made up by tyrosine
5
Biological value of some proteins
Protein
Egg white
Whey
Whole egg
Casein
Beef
Pork
Oats
Wheat flour
Beans
Gelatine
BV (%)
100
100
96
80
77
70
60
53
46
25
6
Whey
• a by-product at (cottage) cheese production
• yellowish liquid (the colour comes from riboflavin)
• cca 12 % of high quality proteins (lactoalbumin, lactoglobulins)
• rich in other B-complex vitamins and lactose
• dried whey is available in shops (esp. fitness centres)
7
Catabolic pathways of AA
•
dietary proteins  AA (GIT, pepsin, trypsin etc.)
•
transamination of AA in cells  glutamate
•
dehydrogenation deamination of glutamate  NH3
•
detoxication of ammonia  urea, glutamine
blue colour indicates the pathway of nitrogen
8
Q. (p. 54)
Write a general equation of a reaction catalyzed
by aminotransferases.
9
General scheme of transamination
R CH COOH
+
NH2
O
2-oxoglutarát
2-oxoglutarate
aminokyselina
aminoacid
aminotransferasa
aminotransferase
pyridoxalfosfát
reversible
reaction
R C
HOOC C CH2CH2COOH
COOH
O
2-oxoacid
2-oxokyselina
+
HOOC CH CH2CH2COOH
NH2
glutamát
glutamate
10
Q. (p. 54)
Which cofactor is used by aminotransferases?
11
Pyridoxal phosphate
R
CH
COOH
NH 2
H
C
HO
O
O
CH 2O P
OH
OH
H 3C
N
12
aminoacid  oxoacid
1. Phase of transamination
pyridoxal-P  pyridoxamine-P
aminokyselina
aminoacid
oxokyselina
2-oxoacid
R CH COOH
R CH COOH
NH2
H
C
O
pyridoxal-P
N
-
R C
rearrangement
izomerace
COOH
N
CH
CH2
Schiff’s base
Schiffova báze
aldimin
pyridoxalu
(aldimine)
Schiff’s base
iminokyselina
(ketimine)
ketimin
oxokyseliny
H2O
H2O
R C
COOH
O
CH2NH2
pyridoxamin-P
13
2. Phase of transamination 2-oxoglutarate  glutamate
pyridoxamine-P  pyridoxal-P
glutamát
glutamate
2-oxoglutarate
2-oxoglutarát
COOH
CH2
CH2
COOH
COOH
CH2
CH2
CH2
CH2
CH2
CH2
HOOC C
HOOC C
O
COOH
-
H2O
N
N
CH2
CH
CH2NH2
pyridoxamin-P
HOOC CH
HOOC CH
H2O
ketiminbase
Schiff’s
aldimin
Schiff’s
base
ketimine
aldimine
oxokyseliny
pyridoxalu
NH2
H
C
O
pyridoxal-P
14
In transaminations, nitrogen of most
AA is concentrated in glutamate
Glutamate then undergoes
dehydrogenation deamination
and releases free ammonia NH3
15
Dehydrogenation deamination of glutamate
is a reversible reaction
GMD
HOOC CH CH2CH2COOH
NH2
HOOC C CH2CH2COOH
- 2H
NH
NAD(P)+
glutamát
glutamate
2-iminoglutarate
2-iminoglutarát
H2O
main source of
ammonia in cells
NH3 + HOOC C CH2CH2COOH
O
2-oxoglutarát
2-oxoglutarate
16
GMD = glutamate dehydrogenase
Intracellular localization
Transamination  glutamate
cytosol
mitochondria
NH3
urea
synthesis
GMD
glutamate
cytosol
Glu + NH3  Gln
17
Q.
What are two main sources of ammonia
in human body?
18
Two main sources of ammonia in body
• Dehydrogenation deamination of glutamate
in cells of most tissues
!
• Bacterial fermentation of proteins in large intestine
ammonia diffuses freely into portal blood

portal blood has high concentration of NH3 
!
NH3 is eliminated by liver (under normal cond.)
19
Other ways of deaminations
• deamination of glutamate in purine nucleotide cycle (p. 55)
• oxidative deamination of some AA ( H2O2)
• oxidative deamination of biogenous amines ( H2O2)
• desaturation deamination of His  urocanic acid + NH3
• oxidative deamination of lysinu
lysyloxidase (Cu2+): Lys + O2  NH3 + allysine + H2O (p. 114)
20
Oxidative deamination of some AA
H2O
R CH COOH
R
NH2
aminoacid
C
COOH
R
COOH
O
NH
FAD
C
FADH2 iminokyselina
iminoacid
oxoacid
NH3
H2O + O2
katalasa
catalase
H2O2
O2
• less frequent
• for glycine
• D-AA
21
Oxidative deamination of amines
monoamino
oxidase
monoaminoxidasa
R CH2
NH2
biogenníamine
amin
biogenous
FAD
R CH NH
FADH2
H2O
R CH O
aldehyd
aldehyde
imin
NH3
H2O2
O2
acid
22
Desaturation deamination of histidine
N
N
CH CH COOH
H
N
H
NH2
- NH3
CH CH COOH
H
N
urocanic acid
23
Other sources of ammonia in tissues
• non-enzymatic carbamylation of proteins
Prot-NH2 + NH2-CO-NH2  NH3 + Prot-NH-CO-NH2
• catabolism of pyrimidine bases
cytosine/uracil  NH3 + CO2 + β-alanine
thymine  NH3 + CO2 + β-aminoisobutyrate
• hem synthesis
4 porfobilinogen  4 NH3 + uroporfyrinogen
24
Three ways of ammonia detoxication
Feature
Urea
Glutamine (Gln)
Glutamate (Glu)
Relevance



Compound type
H2CO3 diamide
γ-amide of Glu
α-aminoacid
Reaction
urea cycle
Glu + NH3
hydrog. amin. 2-OG
Enzyme
5 enzymes
Gln-synthase
GMD
3 ATP
1 ATP
1 NADHa
mitoch. + cytosol
cytosol
mitochondria
only liver
liver + other
mainly brain
Energy needs
Intracell localiz.
Organ localiz.
a
Equivalent of 3 ATP (compare respiratory chain).
25
Hydrolysis of arginine provides urea
CH2CH2CH2CHCOOH
NH2
NH
H2O
CH2CH2CH2CHCOOH
NH2
NH2
ornitin
ornithine
C N H
NH2
NH2
arginine
arginin
O
C
NH2
urea
in urea cycle
26
What is metabolic origine of N atoms in urea?
O
H2N
C
NH2
27
O
H2N
free ammonia
C
NH2
aspartate
(in urea cycle)
28
The properties of urea
• carbonic acid diamide
• perfectly soluble in water
• non-electrolyte  neutral aq. solutions (pH = 7)
• produced in liver × excreted by kidneys
• difuses easily through all cell membranes
29
Glutamine synthesis requires ATP
ATP
ADP + P
COOH
COOH
H2N CH
CH2
CH2
O
C
OH
H2N CH
CH2
+ NH3
CH2
- H2O
glutamine synthetase
glutamate
glutamát
O
C
NH2
glutamine
glutamin
2nd way of NH3 detoxication
30
Compare two reactions (GMD)
dehydrogenation
deamination
of glutamate
+
NADH + H
COOH
NAD
H2N CH
+
NH3
COOH
HN C
H2O
COOH
O C
CH2
CH2
CH2
CH2
CH2
CH2
COOH
COOH
COOH
hydrogenation
amination
of 2-oxoglutarate
3rd way of NH3 detoxication
31
Glucose-alanine cycle
liver
muscle
glucose
glucose
glycolysis
gluconeogenesis
pyruvate
transamination
alanine
transport in blood
pyruvate
transamination
alanine
32
Glutamine cycle
• Gln carries –NH2 group from muscles to liver (periportal
hepatocytes) for detoxification
• in perivenous hepatocytes, Gln is made from glutamate to
keep ammonia concentration low (Glu + NH3  Gln)
• Gln is exported from the liver to kidney where is
deaminated, NH4+ ions are excreted by urine
• exogenous and endogenous Gln is the source of energy
for intestinal mucosa: Gln  2-OG  energy (CAC)
33
Glutamine deamination in kidney occurs stepwise
glutaminase
glutaminasa
Gln
glutamate
dehydrogenase (GMD)
glutamátdehydrogenasa
Glu
NH 3
+
H
NH 4
+
2-oxoglutarate
2-oxoglutarát
NH 3
+
H
NH 4
+
urineMoč
(pH ~ 5)
34
Q.
What reaction is catalyzed by glutaminase?
35
Glutaminase catalyses the hydrolysis of amide group in
glutamine
COOH
COOH
H2N CH
H2N CH
CH2
O
H2O
CH2
CH2
CH2
C
C
NH2
glutamin
glutamine
O
+ NH3
OH
glutamate
glutamát
36
Test results will be available on line
is.muni.cz
Spring 2006
Biochemistry II-s
Notebook
37