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Amino Acid Metabolism. 生化教研室:牛永东. 生物化学的学习方法. 特点一:与数理化不同,尚未进入 定量科学 的阶段,还处在 定性科学 阶段。因此不可能通过公式或定理推出一个准确的结论; 特点二: 是没有绝对,几乎所有的结论都可以被一些例外打破(生物多样性)。 一般性结论:生物化学的学习应以概念为主 --- 以 记忆 为主,在记忆的基础上 加以理解 。. 【 目的与要求 】. 掌握脱氨基作用、 氨的来源、去路 ,氨的转运 …… 掌握尿素合成的部位和全过程 掌握 一碳单位 的概念、来源、载体和功能

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amino acid metabolism

Amino Acid Metabolism

生化教研室:牛永东

slide2
生物化学的学习方法

特点一:与数理化不同,尚未进入定量科学的阶段,还处在定性科学阶段。因此不可能通过公式或定理推出一个准确的结论;

特点二: 是没有绝对,几乎所有的结论都可以被一些例外打破(生物多样性)。

一般性结论:生物化学的学习应以概念为主---以记忆为主,在记忆的基础上加以理解。

slide3
【目的与要求】
  • 掌握脱氨基作用、氨的来源、去路,氨的转运……
  • 掌握尿素合成的部位和全过程
  • 掌握一碳单位的概念、来源、载体和功能
  • 需要掌握的概念:

必需氨基酸、蛋白质的互补作用、氨基酸库、联合脱氨基作用.……

metabolism
Metabolism
  • consists of both catabolic and anabolic processes
  • Catabolism comprises all processes, in which complex molecules are broken down to simple ones
  • Anabolism means any constructive metabolic process by which organisms convert substances into other components required for the organism's chemical architecture
slide5
Introduction
  • Amino acids (AAs) are the building blocks of proteins (precursors for proteins) (物质代谢)
  • Energy metabolites (17.9KJ/g Pr):When degraded, amino acids produce glucose/carbohydrates and ketone bodies(能量代谢)
  • Precursors for many other biological N-containing compounds ,Involved as direct neurotransmitters or as precursors to neurotransmitters, eg. (信息分子代谢)
  • - Tyrosine gives DOPA and dopamine
  • - Precursors to peptide hormones and thyroid hormone
  • - Precursors to histamine, NAD and other compounds of biological importance
slide6
some major biological functions
  • Detoxification of drugs, chemicals and metabolic by-products
  • * Excess dietary AAs are neither storednor excreted. Rather, they are converted to common metabolic intermediates
outline
outline

1. The nutrition of protein

2. The digestion、absorption and putrefaction of protein

3. The general metabolism of AA

4. Metabolism of ammonia

5. Single AA metabolism

section 1 the nutrition of protein
Section 1 The nutrition of protein
  • Nitrogen balance
  • The requirements
  • Classification of amino acids
slide9
Nitrogen balance
  • Zero or total nitrogen balance:
  • the intake = the excretion (adult)
  • Amount of nitrogen intake is equal to the amount of nitrogen excreted is zero or total nitrogen balance
  • Positive nitrogen balance:
  • the intake > the excretion
  • during pregnancy, infancy, childhood and recovery from severe illness or surgery
  • Negative nitrogen balance:
  • the intake < the excretion
  • following severe trauma, surgery or infections. Prolonged periods of negative balance are dangerous and fatal if the loss of body protein reaches about one-third of the total body protein
slide10
The requirements
  • The requirements of protein for the health: the minimal requirement of protein is 30~50 gram for the adult
  • Advice: 80 gram/day (中国营养学会) ? ? ?
slide11
Classification of amino acids
  • non-essential amino acids
  • - can be synthesized by an organism
  • - usually are prepared from precursors in 1-2 steps
  • Essential amino acids ***
  • - cannot be made endogenously
  • - must be supplied in diet
  • eg. Leu, Phe…..
slide12
借来一两本淡色书

*The amino acidsArg, Hisare considered “conditionally essential” for reasons not directly related to lack of synthesis and  they are essential  for growth only

slide13
nutritional value
  • Legumes(豆类) poor in Trp, but rich in Lys;
  • Cereals (谷类) poor in Lys, but rich in Trp
  • Mutual complementation of amino acids
  • Protein deficiency-kwashiorkor, generalized edema and liver enlargement, abdomen bulged
  • Suggestion: the combined-action of protein in diet
slide15
Digestive Tract of protein
  • Proteins are generally too large to be absorbed by the intestine and therefore must be hydrolyzed to the amino acids
  • The proteolytic enzymes responsible for hydrolysis are produced by three different organs: the stomach、pancreas and small intestine (the major organ)
slide16
Stomach
  • HCl (parietal cells ) and Pepsinogen (chief cells )
  • The pH of gastric juice is around 1.0. Food is retained in the stomach for 2-4 hrs
  • HCl kills microorganisms, denatures proteins, and provides an acid environment for the action of pepsin
  • Autocatalysis: pepsinogen is converted to active pepsin(Pepsin A) by HCl
  • Pepsin coagulates milk in presence of Ca2+ ions
slide17
Pancreas and small intestine
  • Endopeptidase(pancreas)
  • Trypsin:carbonyl of arg and lys
  • Chymotrypsin: carbonyl of Trp, Tyr, Phe, Met, Leu
  • Elastase: carbonyl of Ala, Gly, Ser
  • Exopeptidase(pancreas)
  • Carboxypeptidase A:amine side of Ala, Ile, Leu, Val
  • Carboxypeptidase B: amine side of Arg, lys
  • Aminopeptidase (small intestine):
  • cleaves N-terminal residue of oligopeptidaes
  • Dipeptidase (small intestine)
slide18
carboxypepidase

endopeptidase

aminopeptidase

dipeptidase

Amino acids+

1/3

Amino acids

95%

slide19
absorption
  • There is little absorption from the stomach apart from short- and medium- chain fatty acids and ethanol
  • Under normal circumstances, the dietary proteins are almost completely digested to their constituent amino acids, and these end products of protein digestion are rapidly absorbed from the intestine into the portal blood
slide20
Amino acids are transported through the brush border by the carrier protein and it is an active transport
  • The classification of carrier protein:

aciditic; basic; neutral and gly-carrier

2. -glutamyl cycle (-谷氨酰基循环)

3. The bi-and tri- peptidase carrier system in the intestinal mucosa cell

slide21
K+-ATPase

Na+

Amino acids

Carrier protein

ATP

Na+

Amino acids

The mechanism of AA’s absorption

K+

Na+

outer

Member

innner

ADP+Pi

K+

Na+

intestine

slide22
-Gln

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

Cys-Gly

peptidase

-GGT

ATP

AA

Cys

Gly

ADP+Pi

Glu

GSH

ATP

-glutamylcysteine

ADP+Pi

ATP

-glutamyl cycle

  • -gltamyl

cyclotransferase

5oxoprolinase

membrane

GCS synthetase

ADP+Pi

GSH synthetase

slide23
–CO2

Amines

AA

Putrefaction

  • Putrefaction: the process of decay of un-digestive and un-absorbed protein and the products by bacterial, fungal in the intestine 5%

1.Amines(胺):

False neurotransmitter are similar with neurotransmitter

slide24
diffuseblood

Ammonia

intestine bacteria

+NH3

AA

2. Ammonia(氨)-1:

A. some amino acids are degraded by the in the intestine bacteria

slide25
urea

CO2 +2NH3

diffuse blood

Enter instein

ammonia

2. Ammonia(氨)-2:

B. urea from the blood to the intestine with resultant increased diffusion of NH3 into the intestinal

Urea enzyme

Urea in blood

slide26
3. The other toxic material:
  • phenol, indole, sulfureted hydrogen……
section 4 the general metabolism of aa
Section 4 The general metabolism of AA
  • Protein and amino acid turnover
  • Degradation of Amino Acids (Fate of amino group)
  • The metabolism of α-ketoacid
slide28
Body protein

Reutilization for new protein synthesis

Protein degradation

Amino acids

Protein turnover

Protein and amino acid turnover

1-2%

75-80%

T1/2 ?

(half time)

slide29
introduction

1. Proteins constantly being synthesized and degraded - need constant supply of amino acids

- need to degrade to protect from abnormal proteins

- regulate cellular processes

slide30
2. Degraded by ubiquitin label

- Ubiquitin binds lysine side chain

- Targets for hydrolysis by proteosomes in cytosol and nucleus

- ATP required

3. Degraded by the protease and the peptidase in the Lysosome

- non- ATP required

- the hydrolysis-selective are bad

slide31
E2-S-

E1-S-

The ubiquitin degradation pathway

ATP AMP+PPi

E3

E2-SH

(ubiquitin)

E1-SH

E2-SH

E1-SH

E1:activiting enzyme E2:carrier protein E3:ligase

ubiquitinational protein

ATP

19S regulate substrate

ATP

20S Proteasome

阿龙-西查诺瓦

26S Proteasome

slide32
Diet protein

Nonprotein nitrogen derivatives

Tissue protein

transamination

Carbohydrate

(glucose)

Ketone dodies

Acetyl-CoA

Amino nitrogen in glutamate

Citric

Acid

Cycle

deamination

NH3

Urea

CO2

Amino acid pool

Overview of the protein metabolism

slide33
-

COO

+

a

H N

3

H

Degradation of Amino Acids

- Reactions in amino acid metabolism

Amino acid

Carboxylic group

Amino group

R group

slide34
introduction
  • Free amino acids are metabolized in identical ways, regardless of whether they are released from dietary or intracellular proteins
  • The metabolism of the resulting amino group and nitrogen excretion are a central part of nitrogen metabolism
slide35
FATE OF AMINO GROUP

DEAMINATION

A. Transamination

B. Oxidative deamination

C. purine nucleotide cycle

a transamination
A. Transamination
  • Transamination by Aminotransferase (transaminase)
  • always involve PLP coenzyme (pyridoxal phosphate)
  • reaction goes via a Schiff’s base intermediate
  • all transaminase reactions are reversible
slide37
Aminotransferases
  • Aminotransferases can have specificity for the alpha-keto acid or the amino acid
  • Aminotransferases exist for all amino acids except proline and lysine
  • The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and a-ketoglutarate, which participate in reactions with many different aminotransferases
    • to an alpha-keto acid alpha-amino acid
slide38
Transamination

aminotransferases

slide39
glutamate-pyruvate aminotransferase

GPT, ALT

Glu+pyruvate

-Ketoglutarate+Ala

(丙酮酸)

(α-酮戊二酸)

Glutamic oxaloacetictransaminase

GOT, AST

Glu+Oxaloacetate

-Ketoglutarate+Asp

(草酰乙酸)

*** ALT and AST are components of a "liver function test". Levels increase with damage to liver (cirrhosis, hepatitis) or muscle (trauma)

slide40
–H2O

+

+H2O

AA

PLP

Schiff’s base

The mechanism of transamination

slide41
Molecule rearrange

Schiff’s base isomer

+H2O

+

–H2O

-ketoacid

PMP(磷酸吡哆胺)

transamination
Transamination
  • Interconversion of amino acids
  • Collection of N as glu
  • Provision of C-skeletons for catabolism
slide43
B. Oxidative Deamination
  • L-glutamate dehydrogenase (in mitochondria)
  • Glu + NAD+ (or NADP+) + H2O NH4+ + a-ketoglutarate + NAD(P)H +H+
  • Requires NAD+ or NADP + as a cofactor
  • Plays a central role in AA metabolism ?
slide44
urea cycle

?

It is inhibited by GTP and ATP, and activated by GDP and ADP

slide46
Combined deamination =

Transamination + Oxidative Deamination

The major pathway !!!

slide47
NH3

AA

Asp

IMP

-Keto

glutarate

H2O

AST

aminotransferases

C. purine nucleotide cycle

AMP

-Keto

acid

Oxaloacetate

fumarate

malate

slide48
The metabolism of α-ketoacid
  • Biosynthesis of nonessential amino acids
  • TCA cycle member + amino acid α-keto acid + nonessential amino acid
  • A source of energy (10%) ( CO2+H2O )
  • Glucogenesis and ketogenesis
slide49
* Classification of amino acids
  • * glucogenic amino acid : are converted into either pyruvate or one of the citric acid cycle intermediates
  • (a-ketoglutarate, succinyl CoA, fumarate or malate)
  • * ketogenic amino acid: will be deaminated via Acetylc-CoA and thus can be made into a ketone body. such as: Leucine and lysine
  • * glucogenic and ketogenic amino acid: isoleucine, phenylalanine, tryptophan and tyrosine, threonine
slide51
1. amino acids degradation

MAO

RCCO2H2NH2 RCHO+NH3

Monoamine oxidase

*** Sources

2. glutamine (glutaminase, kidney)

3. catabolism from bacteria in intestine (two)

4. purine and pyrimidine catabolism

metabolism of ammonia
Metabolism of ammonia
  • Fix ammonia onto glutamate to form glutamine and use as a transport mechanism
  • Transport ammonia by alanine-glucose cycle and Gln regeneration
  • Excrete nitrogenous waste through urea cycle
slide53
Transport of ammonia
  • alaninie - glucose cycle *
  • regenerate Gln
slide54
Alanine-Glucose cycle
  • In the liver alanine transaminase tranfers the ammonia to α-KG and regenerates pyruvate. The pyruvate can then be diverted into gluconeogenesis. This process is refered to as the glucose-alanine cycle
slide56
**** Urea synthesis
  • Synthesis in liver (Mitochondriaandcytosol)
  • Excretion via kidney
  • To convert ammonia to urea for final excretion
slide57
The urea cycle:1932 by Hans Krebs and Kurt Henseleit as the first metabolic cycle elucidated

arginase

Ornithine cycle

Krebs-henseleit cycle

slide58
1

OCT

Mitochondria

2

(鸟氨酸)

(瓜氨酸)

5

3

cytosol

4

slide59
UREA CYCLE (liver)

1. Overall Reaction:

NH3 + HCO3– + aspartate + 3ATP + H2O  urea + fumarate + 2 ADP + 2 Pi + AMP + ppi

2. Requires 5 enzymes:

2 from mitochondria and 3 from cytosol

slide60
Regulation of urea cycle

1.Mitochondrial carbamoyl phosphate synthetase I (CPS I)

CPS I catalyzes the first committed step of the urea cycle

   CPS I is also an allosteric enzyme sensitive to activation by N-acetylglutamate(AGA)which is derived from glutamate and acetyl-CoA

slide61
Increased rate of AA degradation requires higher rate of urea synthesis

 AA degradation ↑glutamate concentration →↑synthesis of N-acetylglutamate ↑CPS I activity ↑urea cycle efficiency

slide62
2. All other urea cycle enzymes are controlled by the concentrations of their substrates

Deficiency in an E ↑(substrate) ↑rate of the deficient E

3. The intake of the protein in food

the intake ↑ ↑urea synthesis

slide63
Hyper-ammonemia and the toxic of the ammonia
  • Hyperammononemia: ammonia intoxication - tremors, slurring of speech, and blurring of vision, coma/death
  • Cause by cirrhosis of the liver or genetic deficiencies
section 5 single aa metabolism
Section 5 Single AA metabolism
  • Decarboxylation

some neurotransmitters’ precursors for the decarboxylation of AAs production– bioactive amines

slide65
L-Glu decarboxylase

– CO2

GABA

L-Glu

-aminobutyric acid (GABA)

Glutamine can be decarboxylated in a similar PLP-dependent fashion, outputting -aminobutyric acid (neurotransmitter, GABA)

slide66
Decarboxylase

3(O)

-CO2

taurine

L-Cys

Taurine

L-cysteinecanbe decarboxylated and converted into outputted the taurine

slide67
Histidine decarboxylation

– CO2

L-Histidine

Histamine

Histamine

Histidinecan also be decarboxylated in a similar PLP-dependent fashion, outputting the Histamine

slide68
Tryptophan

Hydroxylase

decarboxylase

– CO2

5-hydroxytryptophan(5-HT)

5-HT

slide69
Polyaminies: (putrescine,spermidine,spermine)

CO2 is then cleaved off in a PLP-dependentdecarboxylation, resulting in the polyaminies (such as: SAM, spermidine, spermine)

one carbon unit metabolism
***** One carbonunit metabolism
  • One carbon unit: some AA may turn into the group including one carbon in the AA metabolism

Such as:

methl -CH3 甲基

methylene -CH2 亚甲基/甲烯基

methenyl -CH= 次甲基/甲炔基

formyl -CHO 甲酰基

formimino -CH=NH 亚氨甲基

But without CO2

slide72
One carbon unit metabolism
  • Folic acid / folate is an essential vitamin, and as such it cannot be synthesized within the human body
  • Folate itself is not an active cofactor; its doubly-reduced form, is tetrahydrofolate (THF)
  • Tetrahydrofolate (THF) : the carrier of one carbon
  • accepts one carbon groups from amino acids
slide73
Folate is reduced first by dihydrofolate reductase (DHFR) into dihydrofolate (DHF), oxidizing an NADPH in the process. DHFR, again oxidizing an NADPH+H+/NADP+, can also reduce DHF into THF
slide74
10

10

5

5

( N5-CH3-FH4)

(N5,N10-CH2-FH4)

(Folate)

slide75
THF – biosynthetic pathways
  • There are three important biosynthetic precursors synthesized from THF:
  • N5,N10-methylene-THF
  • N5-methyl-THF
  • N10-formyl-THF
  • N5,N10-methylene-THF acts in a central processing role in the synthesis of all of these enzymes: in order to synthesize either of the other two, one must first produce N5,N10-methylene-THF

N5,N10甲烯四氢叶酸

slide76
Serine Hydroxymethyl Transferase

Serine

NAD+

NADH

NADP+

Glycine

NADPH

CO2+ NH3

biosynthesis

methionine

Histidine

N5-methyl-THF

THF

biosynthesis

thymidylate

N5,N10-methylene-THF

N5,N10甲烯四氢叶酸

NAD+

THF

N5,N10-methenyl-THF

N5,N10甲炔四氢叶酸

Glycine

cataboase

H2O

biosynthesis

purines

NADH

N10-formyl-THF

Tryptophan

slide77
Sulfanilamide (磺胺) is a compound that the human body can create. It is a close analog to PABA, and it has the effect of stopping folate synthesis in bacteria. There are a wide variety of “sulfa drugs ” (磺胺类药) based on PABA analogs such as sulfanilamide
  • Trimethoprim(TMP) and pyrimethamin(百炎净), on the other hand, are DHFR inhibitors. Because bacterial DHFR is structurally simpler than human DHFR, these two drugs have a more drastic effect on bacteria than they do on us
slide78
S-CH

—S——S—

CH

SH

CH

CH

3

2

2

2

(CH

)

CHNH

CHNH

CHNH

2

2

2

2

2

CH-NH

COOH

COOH

COOH

2

COOH

Metabolism of sulfur-containing amino acids

cysteine

cystine

Met

slide79
Methionine Catabolism
  • The principal fates of methionine are incorporation into polypeptide chains(protein synthesis), and use in the production of a-ketbutyrate and cysteine via S-adenosyl methionine (SAM)
slide80
+

Adesine transferase

– PPi +Pi

SAM

Met

ATP

S-adenosyl methionine (SAM)

  • is a powerful methylating agent (in the methylatinggene regulationof DNA and RNA, It is constantly regenerated in a cyclical) with uses in many biochemical pathways
  • formed from ATP and Met
s adenosylmethionine sam
S-adenosylmethionine (SAM)
  • methyl group is donated to form several products

norepinephrine --> epinephrine

gamma-butyric acid --> carnitine

guanidinoacetate --> creatine

(胍乙酸)

slide82
SAM

Met

PPi +Pi

ATP

COOH

RH

FH4

N5-CH3-FH4

methyl-transferase

H

C-NH

2

N5-CH3-FH4

(CH

)

2

2

A

R-CH3

H2O

R

CH

S

2

O

SAH

O

H

O

H

Met cycle

FH4is produced!!!

SAHH

同型半胱氨酸

S-腺苷同型半胱氨酸

slide83
N5,N10-methylene-FH4

NADH

Carbon donors (serine, glycine) combine with THF

NAD+

FH4

N5-CH3-FH4

Only one!

同型半胱氨酸

homocysteine

methionine

vitamin B12

adenosine

ATP

FH4 and Met cycle

all 3 phosphate groups are lost!

H2O

PPi + Pi

s-adenosylhomocysteine

s-adenosylmethionine

S-腺苷同型半胱氨酸

methyl group donated to biological substrate, e.g. norepinephrine

slide84
Regulation of the Met metabolism
  • If methionine and cysteine are present in adequate quantities, SAM accumulates and is a positive effector on cystathionine synthase(胱硫醚合成酶), encouraging the production of cysteine and a-ketobutyrate
  • If methionine is scarce, SAM will form only in small quantities, thus limitingcystathionine synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N5-methyl THF and other compounds as methyl donors
slide85
(胍乙酸)

Creatine metabolism

H2O

slide86
—S——S—

– 2H

2

cysteine

cystine

Metabolism of Cystine and Cysteine

+ 2H

slide87
Cysteine Catabolism
  • The pathway is catalyzed by a liver desulfurase and produces pyruvate and hydrogen sulfide (H2S)
  • The enzyme sulfite oxidase uses O2 and H2O to convert HSO3- to sulfate (SO4-) , and H2O2. The resultant sulfate is used as a precursor for the formation of 3'-phosphoadenosine-5'-phosphosulfate, PAPS
slide88
Metabolism of aromatic amino acid

Phenylalanine

Tyrosine

tryptophan

slide89
Phenylalanine metabolism

Phenylalanine hydroxylase

THF

DHF

NADP+

NADPH+H+

tyrosine

phenylalanine

slide90
Tyr metabolism

1. Catecholamine/melanin

slide91
NH3+

|

- CH2 - CH - COO-

NH3+

|

- CH2 - CH - COO-

O

||

- CH2 - C - COO-

Homogentisate

Phe

Hydroxylase

Tyr

HO -

HO -

Tyr

aminotransferase

HO - - OH

CH2 - COO-

TyrCatabolism

尿黑酸

对羟基丙酮酸

slide92
Homogentisate

-OOC - CH = CH - C - CH2 - C - CH2 - COO-

|| ||

O O

cis

trans

-OOC - CH = CH - C - CH2 - C - CH2 - COO-

|| ||

O O

fumarate

O

acetoacetate

H ||

-OOC - C = C - COO- + H3C - C - CH2 - COO-

H

HO - - OH

CH2 - COO-

尿黑酸

乙酰乙酸

延胡索酸

slide93
Phenylalanine Hydroxylase & PKU
  • Phenylketonuria (PKU) = lack of phenylalanine hydroxylase
  • - can’t hydroxylate phenylalanine to tyrosine

[Phe] = 0.1 mM normally

 1.2 mM in PKU

1 in 20,000 homozygous

1 in 150 heterozygous

IQ study:

53  93

slide94
People with phenylketonuria must avoid excess phenylalanine, but both tyrosine and phenylalanine are essential amino acids, so they shouldn’t exclude it completely or brain disorders with result
slide96
Branched-chain amino acids (BCAAs):

isoleucine, leucine and valine

  • The catabolism of all three BCAAs initiates in muscle and yields NADH and FADH2 which can be utilized for ATP generation
slide97
Isoleucine / Leucine / Valine

-ketoglutarate

transamination

A metabolic block here causes maple syrup urine disease

glutamate

-keto acid

NAD+, CoASH

-keto acid dehydrogenase

NADH, CO2

gluconeogenesis

-keto-S-CoA

(if Leu or Lys, only

this path can be used)

Succinyl-CoA

Proprionyl-CoA

ketogenesis

丙酰CoA

slide98
Most nitrogen metabolism pathways are very complex

require many steps

require input of ATP and NADPH

are regulated by feedback inhibition mechanisms (allosteric)

ad