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 • 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• 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

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

some major biological functions

• Detoxification of drugs, chemicals and metabolic by-products

* Excess dietary AAs are neither stored nor excreted. Rather, they are converted to common metabolic intermediates

outline1. The nutrition of protein2. The digestion 、 absorption and

putrefaction of protein3. The general metabolism of AA4. Metabolism of ammonia5. Single AA metabolism

Section 1 The nutrition of protein

• Nitrogen balance• The requirements• Classification of amino acids

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

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 ) ? ? ?

• 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…..

Classification of amino acids

Nonessential EssentialAlanine Arginine*

Asparagine Histidine *Aspartate ValineCysteine Lysine

Glutamate IsoleucineGlutamine Leucine

Glycine Phenylalanine Proline MethionineSerine Threonine

Tyrosine Tyrptophan *The amino acids Arg, His are considered “conditionally essential” for reasons not directly related to lack of synthesis and they are essential for growth only

借来一两本淡色书

nutritional value• Legumes(豆类 ) poor in Trp, but rich in Lys; Cereals (谷类谷类 ) poor in Lys, but rich in Trp • Mutual complementation of amino acids • Protein deficiency-kwashiorkor, generalized ed

ema and liver enlargement, abdomen bulged • Suggestion: the combined-action of protein

in diet

Section 2The digestion 、 absorption and putrefaction 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)

Digestive Tract of protein

Stomach• HCl (parietal cells ) and Pepsinogen (chief cells

) • The pH of gastric juice is around 1.0. Food is re

tained 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

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)

NH2 CH C NH CH- -

R1

O

R2

-NH CH C NH CH- -

R3

O

R4

-NH CH C NH CH

R5

O

R6

COOH

aminopeptidase

endopeptidase carboxypepidase

NH2 CH C NH CHO

R"

COOHR'

Amino acids +

Amino acids

dipeptidase 1/3

95%

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

• Amino acids are transported through the brush border by the carrier protein and it is an active transport

1. 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 th

e intestinal mucosa cell

ADP+Pi

ATP

K+

K+ Na+

Na+

Na+Amino acids

Na+Amino acidsouter

Member

innner

K+-ATPase

Carrier protein

The mechanism of AA’s absorption

intestine

Cys-Gly

-gltamyl cyclotransferase5oxoprolinase

GSH synthetase

GCS synthetase membrane

-glutamyl cycle

Gly Cys

peptidase

GSH

-GGT

|||||||||||

|||||||||||

AA

COOHCHR

H2N

-GlnCOOHCHNH2CH2CH2CO

HN CH

COOH

R

COOHCHR

H2N

ATP

ATP

ATP

ADP+Pi

Glu ADP+Pi

-glutamylcysteineADP+Pi

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

Putrefaction

1.Amines(胺 ):–CO2

Amines

RCH2NH2

AA

CH COOHR

NH2

False neurotransmitter are similar with neurotransmitter

Ammonia

diffuse blood

intestine bacteria

+ NH3CH2 COOHR

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

AA

CH COOHR

NH2

2. Ammonia( 氨 )-1:

C=O

NH2

NH2

urea

Enter instein

Urea enzyme CO2

+2NH3

diffuse blood

ammoniaUrea in blood

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

2. Ammonia( 氨 )-2:

3. The other toxic material: phenol, indole, sulfureted hydrogen…

…

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

Body protein

Amino acids

Protein degradation

Protein turnover

Reutilization for new protein synthesis

Protein and amino acid turnover

1-2%

75-80%

T1/2 ?(half time)

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

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

The ubiquitin degradation pathwayE1-E1-S-S-

E1-E1-SHSH

E2-E2-S-S-E1-E1-

SHSH

E2-E2-SHSH

E2-E2-SHSH

ATP AMP+PPiATP AMP+PPi EE33

ubiquitinational protubiquitinational proteineinATPATP

26S Proteasom26S Proteasomee

20S 20S ProteasomProteasomeeATPATP

19S regulate substra19S regulate substratete

E1 ： activiting enzyme E2 ： carrier protein E3 ： ligase

（ ubiquitin ）

阿龙 - 西查诺瓦

Amino acid pool

Diet protein

Tissue protein

Carbohydrate(glucose)

transamination Nonprotein nitrogen derivatives

Amino nitrogen in glutamate

deamination NH3

Urea

Acetyl-CoA

CitricAcidCycle

CO2

Ketone dodies

Overview of the protein metabolism

R group

aH N3+ HAmino group

Carboxylic group

COO- Degradation of Amino Acids - Reactions in amino acid

metabolismAmino acid

• 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

introduction

FATE OF AMINO GROUP DEAMINATION A. Transamination B. Oxidative deamination C. purine nucleotide cycle

A. Transamination• Transamination by Aminotransferase

(transaminase)• always involve PLP coenzyme (pyridoxal

phosphate)• reaction goes via a Schiff’s base interm

ediate• all transaminase reactions are reversible

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

Transamination

aminotransferases

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

Glu+pyruvate glutamate-pyruvate aminotransferase GPT, ALT-Ketoglutarate+Ala

Glu+Oxaloacetate Glutamic oxaloacetictransaminase GOT, AST -Ketoglutarate+Asp

（（草酰乙酸草酰乙酸））（（ αα--酮戊二酸酮戊二酸））((丙酮酸丙酮酸））

The mechanism of transamination

+ N

CH3OH

CH2OPO3H2

CO

H

PLP

–H2O

+H2O

Schiff’s base

AA

HOOC C NH2

H

R1

Schiff’s base isomer

N

CH3OH

CH2OPO3H2

CH2NH2

PMP( 磷酸吡哆胺 )–H2O+H2O

Molecule rearrange

-ketoacid+

HOOC C OR1

Transamination• Interconversion of amino acids• Collection of N as glu• Provision of C-skeletons for cataboli

sm

B. Oxidative Deamination• L-glutamate dehydrogenase (in mitochondria) Glu + NAD+ (or NADP+) + H2O NH4+ + a-ketogl

utarate + NAD(P)H +H+

Requires NAD+ or NADP + as a cofactor Plays a central role in AA metabolism ?

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

urea cycle ?

Combined Deamination

?

Combined deamination ＝ Transamination + Oxidative Deamin

ation The major pathway ！！！

AA-Ketoglutarate

-Keto acid

Asp

Oxaloacetatemalate fumarate

IMP

AMP

H2O

NH3

C. purine nucleotide cycle

aminotransferases AST

The metabolism of α-ketoacid• Biosynthesis of nonessential amino acid

s TCA cycle member + amino acid α-k

eto acid + nonessential amino acid • A source of energy (10%) ( CO2+H2O )• Glucogenesis and ketogenesis

* 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

Ammonia is toxic, so cells need to get rid of it…..

1. amino acids degradation

RCCO2H2NH2 RCHO+NH3

MAO

Monoamine oxidase

*** Sources

2. glutamine (glutaminase, kidney)3. catabolism from bacteria in intestine (two) 4. purine and pyrimidine catabolism

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

Transport of ammonia

• alaninie - glucose cycle *

• regenerate Gln

Alanine-Glucose cycleIn 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

Gln regeneration

**** Urea synthesis

• Synthesis in liver (Mitochondria and cytosol) • Excretion via kidney • To convert ammonia to urea for final excreti

on

Ornithine cycle

The urea cycle ： 1932 by Hans Krebs and Kurt Henseleit as the first metabolic cycle elucidated

Krebs-henseleit cycle

arginase

Mitochondria

cytosol

OCT

（瓜氨酸）

（鸟氨酸）

1 2

3 4

5

UREA CYCLE (liver) 1. Overall Reaction: NH3 + HCO3– + aspartate + 3 ATP + H2O ure

a + fumarate + 2 ADP + 2 Pi + AMP + ppi 2. Requires 5 enzymes: 2 from mitochondria and 3 from cytosol

Regulation of urea cycle1.Mitochondrial carbamoyl phosphate synthetase I (CPS I) CPS I catalyzes the first committed step of th

e urea cycle CPS I is also an allosteric enzyme sensitive t

o activation by N-acetylglutamate （ AGA ） which is derived from glutamate and acetyl-CoA

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

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

• Hyperammononemia: ammonia intoxication - tremors, slurring of speech, and blurring of vision, coma/death

• Cause by cirrhosis of the liver or genetic deficiencies

Hyper-ammonemia and the toxic of the ammonia

Section 5 Single AA metabolism

• Decarboxylation some neurotransmitters’ precursors fo

r the decarboxylation of AAs production– bioactive amines

L-Glu decarboxylase– CO2

GABA

(CH2)2

COOH

CH2NH2

L-Glu

(CH2)2

COOH

CHNH2

COOH

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

Decarboxylase-CO2

L-CysCOOH

CHNH2

CH2SH

taurineCHNH2

CH2SO3H3 （ O ）COOH

CHNH2

CH2SO3H

Taurine L-cysteine can be decarboxylated and converted into outputted the taurine

– CO2

Histidine decarboxylation

Histamine

CH2CH2NH2

NHN

L-HistidineCOOHCCH2

NH2NHN

H

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

NH

CH2 CH2NH2OH

5-hydroxytryptophan （ 5-HT ）COOHCHCH2

NH2

NH

Tryptophan– CO2

Hydroxylase decarboxylase

5-HT

Polyaminies: (putrescine,spermidine,spermine)

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

***** One carbon unit metabolism• One carbon unit: some AA may turn into the group includ

ing 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

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

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

5

10

（ N5-CH3-FH4 ）N

NH

NH2 NH

N

CH3

CH2

NH RO

(Folate)

N

NH

NH2 NH

N

ON

R

（ N5 ， N10-CH2-FH4 ）

5

10

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

NADHNAD+

NADPHNADP+

N5,N10-methylene-THF

THF

THF

NAD+

NADH

Glycinecataboase

Serine

Glycine

CO2+ NH3

Serine Hydroxymethyl Transferase

N5-methyl-THF

N5,N10-methenyl-THF

N10-formyl-THF

H2O

biosynthesismethioninebiosynthesisthymidylate

biosynthesispurinesTryptophan

Histidine

N5,N10 甲烯四氢叶酸

N5,N10 甲炔四氢叶酸

• 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

—S——S—

COOHCHNH 2

CH2

COOHCHNH 2

CH2

cysteine cystineMet

(CH2)2

S-CH 3

CH-NH2

COOHCOOHCHNH2

CH2SH

Metabolism of sulfur-containing amino acids

Methionine Catabolism • The principal fates of methionine are incorporati

on into polypeptide chains(protein synthesis), and use in the production of a-ketbutyrate and cysteine via S-adenosyl methionine (SAM)

S-adenosyl methionine (SAM) • is a powerful methylating agent (in the methylating

gene regulation of DNA and RNA, It is constantly regenerated in a cyclical) with uses in many biochemical pathways

• formed from ATP and Met

+R

OO

OHOH

P

O

OH

P

O

O

OH

OH

OH

O

O

P

ATPMet

(CH2)2

S-CH3

CH-NH2

COOH

Adesine transferase– PPi +Pi

SAM

ROCH2

OHOH

S+

(CH2)2

CH3

C-NH2

COOH

H

S-adenosylmethionine (SAM)• methyl group is donated to form several products norepinephrine --> epinephrine gamma-butyric acid --> carnitine guanidinoacetate --> creatine ( 胍乙酸 )

SAM

PPi +PiR

OCH2

OHOH

S+

(CH2)2

CH3

C-NH2

COOH

H Met cycle

N5-CH3-FH4

FH4N5-CH3-FH4methyl-transferase

Met(CH2)2

S-CH3

CH-NH2

COOH ATP

H2OA

SAHHS

(CH2)2

C-NH2

COOH

H

SAHH

FH4 is produced!!!

S-腺苷同型半胱氨酸同型半胱氨酸 OHOH

RH

ROCH2S

(CH2)2

C-NH2

COOH

H

R-CH3

homocysteine methionine

s-adenosylmethionines-adenosylhomocysteine

vitamin B12 ATP

PPi + Pi

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

adenosine

H2O

N5-CH3-FH4 FH4

N5,N10 -methylene- FH4 NADH

NAD+

Carbon donors (serine, glycine) combine with THF

all 3 phosphate groups are lost!

S-腺苷同型半胱氨酸

同型半胱氨酸 Only one!

FH4 and Met cycle

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 limiting cystathionine synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N5-methyl THF and other compounds as methyl donors

H2O

Creatine metabolism

( 胍乙酸 )

+ 2HCOOH

CHNH2

CH2SH

2

cysteine

– 2H

COOH

CHNH2

CH2

COOH

CHNH2

CH2—S——S—

cystine

Metabolism of Cystine and Cysteine

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 c

onvert HSO3- to sulfate (SO4-) , and H2O2. The resultant sulfate is used as a precursor for the formation of 3'-phosphoadenosine-5'-phosphosulfate, PAPS

Metabolism of aromatic amino acid

PhenylalaninePhenylalanine TyrosineTyrosine

CH2

CHNH2

COOH

CH2

CHNH2

COOH

OH

tryptophan

Phenylalanine hydroxylaseTHF DHF

NADPH+H+NADP+

phenylalanine

tyrosine

Phenylalanine metabolism

1. Catecholamine/melanin

Tyr metabolism

NH3+ |- CH2 - CH - COO-

CH2 - COO-

HO - - OH

Homogentisate

NH3+ |- CH2 - CH - COO-HO -

Tyr Phe Hydroxylase

O ||- CH2 - C - COO-HO -

Tyraminotransferase

尿黑酸

对羟基丙酮酸

Tyr Catabolism

CH2 - COO-

HO - - OH Homogentisate

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

|| || O O

cis

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

|| || O O

trans

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

H

O acetoacetatefumarate

尿黑酸

乙酰乙酸

延胡索酸

• Phenylketonuria (PKU) = lack of phenylalanine hydroxylase

- can’t hydroxylate phenylalanine to tyrosine

Phenylalanine Hydroxylase & PKU

[Phe] = 0.1 mM normally 1.2 mM in PKU 1 in 20,000 homozygous1 in 150 heterozygous

IQ study: 53 93

• 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

The Metabolism of Branched Chain Amino

Acids

• Branched-chain amino acids (BCAAs): isoleucine, leucine and valine • The catabolism of all three BCAAs initiat

es in muscle and yields NADH and FADH2 which can be utilized for ATP generation

Isoleucine / Leucine / Valine

-keto acid

-keto-S-CoA

-ketoglutarateglutamate

transamination

NAD+, CoASH

NADH, CO2

-keto acid dehydrogenase

(if Leu or Lys, onlythis path can be used)ketogenesisProprionyl-CoASuccinyl-CoA

gluconeogenesis

A metabolic block here

causes maple syrup urine

disease

丙酰 CoA

• Most nitrogen metabolism pathways are very complex

require many steps require input of ATP and NADPH are regulated by feedback inhibition

mechanisms (allosteric)