5/10/2012

Amino Acid Metabolism

Chem 4220 IAUPR Barranquitas JA Negrn

1

Summary
Introduction to protein metabolism Ubiquitin and protein degradation Amino acid pool Transamination Urea cycle Ketogenic and glucogenic amino acids

JA Negrn, Ph.D.

5/10/2012

Introduction to Protein Metabolism

Anabolism
Biosynthesis of Proteins

Catabolism
Urea cycle Amino acid catabolic pathways The destruction of proteins is as important as their synthesis for the maintenance of protein homeostasis in cells. In eukaryotes, the ubiquitinproteasome system (Nobel Prize in Chemistry 2004) is responsible for most of this protein degradation.
3

JA Negrn, Ph.D.

Ubiquitinproteasome system
It is known that ubiquitin-mediated destruction plays a crucial part in cell-cycle regulation, DNA repair, cell growth and immune function, as well as in hormonemediated signalling in plants. Ubiquitin has been shown to have numerous non-proteolytic functions.

JA Negrn, Ph.D.

5/10/2012

UBIQUITIN

76 Amino Acid polypeptide Highly conserved in evolution: 3 Amino acid differences between yeast and human homologues

JA Negrn, Ph.D.

The Ubiquitin Pathway

Ub Ub Ub

E1

E2

E3

Target
Ub Ub Ub Ub 26s proteosome degradation
6

JA Negrn, Ph.D.

5/10/2012

Ubiquitin Pathway

JA Negrn, Ph.D.

ATP consuming process

JA Negrn, Ph.D.

5/10/2012

Nitrogen
Natural occurring nitrogen, N2, is not usable in biological systems. Only a few organisms can convert N2 to NH3, which is usable by biological systems. Most organisms guard NH3 very closely, however, because of the routine intake of NH3 containing compounds we need a way to rid of it.
9

JA Negrn, Ph.D.

N2 balance

Protein-major dietary source of N2 Amino acids

Protein synthesis N2 containing compounds Excess disposed through urea (80%), ammonia, uric acid, creatinine in urine (very small amount in fecal matter)
10

JA Negrn, Ph.D.

5/10/2012

Amino Acid Pool

Dietary Proteins

Digestion
Plasma Proteins

Amino Acid pool

Deamination

Anabolism

Catabolism

Tissue Proteins

Amino acid derivatives

ketoacid residues (from carbon skeletons of amino acids

Excretion

Energy metabolism in pathways of glucose or fatty acid metabolism JA Negrn, Ph.D.

Urea, ammonia in the urine

11

Amino Acid Pool, more details

12

JA Negrn, Ph.D.

5/10/2012

Essential and Non-essential AAs

Essential AAs-Humans (mammals) cannot synthesize their carbon skeletons de novo. Some EAA are considered essential because we cannot synthesize enough, especially for growth (children). Non-essential AAs-Synthesized from intermediates of glucose/TCA cycle except TYR (cannot make aromatic ring). Conditionally essential
Arg: Not enough His: Essential for growth in children Phe: Necessary to produce Tyr Tyr: Made from Phe (phenylketonuria) Cys: Produce from Met Pro: Made from Glu and ornithine (urea) Gln: critically ill, stress (fuel for proliferative tissues)
13

JA Negrn, Ph.D.

Amino Acid Degradation

Amino Acids

NH3 Urea Cycle

CO2 + H2O

Acetyl CoA

Acetoacetyl CoA
14

JA Negrn, Ph.D.

5/10/2012

Catabolism of amino acids

Amino acid catabolism takes place in the liver. The first step is the removal of the a-amino group by enzymes called amino-transferases or transaminases. The amino group is transferred to aketoglutarate to make glutamate.
15

JA Negrn, Ph.D.

Glutamate
Amino acid catabolism takes place in the liver. The receiver of the amino group from amino acids in the liver is a-ketoglutarate and its product is glutamate. The enzyme is an aminotransferases or transaminases. Pyridoxal phosphate (PLP) or the active form of Vitamin B6 is required by the amino transferase.
16

JA Negrn, Ph.D.

5/10/2012

Glutamate releases its amino group as ammonia in the mitochondria of liver cells by glutamate dehydrogenase (GluDesh), the only enzyme that can use either NAD+ or NADP+ as reducing equivalent receivers.

GluDesh

17

JA Negrn, Ph.D.

Major Functions of Amino Acids Derived from Dietary Protein

Oxidation Glycogenic amino acids: --Blood glucose--Energy Ketogenic amino acids: -Acetyl CoA-Stored fat-Energy Biosynthesis of nitrogen-containing metabolites

Heme Choline Glycosamine Nucleotides Protein synthesis Biogenic amines Carnitine Creatine phosphate

Blood cell PL Sugar DNA Protein Neurotransmitters Heart Energy

18

JA Negrn, Ph.D.

5/10/2012

The different forms of excreted nitrogen

19

Urea Cycle
Urea is the major end product of nitrogen metabolism in humans and mammals. Ammonia, the product of oxidative deamination reactions, is toxic in even small amounts and must be removed from the body. The urea cycle or the ornithine cycle describes the conversion reactions of ammonia into urea.

20

JA Negrn, Ph.D.

10

5/10/2012

Urea
If not used in the production of new amino acids or other nitrogenous compounds, amino groups are transferred to the liver and converted to urea.

Urea is produced in the cytosol via the urea cycle.

Almost all urea is produced in the liver.

21

JA Negrn, Ph.D.

Urea
Urea produced in liver cells then passes into the bloodstream. It then finds its way to the kidneys to be excreted in the urine. Urea is very soluble in water - about 10.5 M at 25 C.
22

JA Negrn, Ph.D.

11

5/10/2012

The Urea Cycle

A five step process. The first two steps take place within the mitochondrion. The remaining three of which take place in the cytosol.

23

JA Negrn, Ph.D.

The enzymes catalyzing the urea cycle reactions

1. ornithine transcarbamoylase 2. argininosuccinate synthetase 3. argininosuccinase 4. arginase

24

12

5/10/2012

The Urea Cycle and TCA Cycle are interconnected

Some enzymes of the TCA cycle have isozymes in the cytosol, fumarase and malate dehydrogenase for example. Fumarate from argininosuccinate can be converted to malate and then to oxaloacetate, some of which can make their way into the mitochondrion and into the TCA cycle. The aspartate-argininosuccinate shunt provides metabolic links between the pathways by which amino groups and carbon skeletons of amino acids are processed.
25

JA Negrn, Ph.D.

26

13

5/10/2012

Regulation of the Urea Cycle

Within an individual the movement of nitrogen through the cycle depends on diet.

Changes in diet will only affect urea cycle activity over the long term.

27

JA Negrn, Ph.D.

Regulation of the Urea Cycle

Short term, allosteric regulation occurs with carbamoyl phosphate synthetase.

N-acetylglutamate activates carbamoyl phosphate synthetase, and

Arginine activates N-acetylglutamate synthase, which catalyzes the production of Nacetylglutamate, so arginine also is an indirect activator of carbamoyl phosphate synthetase.
28

JA Negrn, Ph.D.

14

5/10/2012

Energetic cost of The Urea Cycle

2NH4+ + HCO3- + 3ATP + H2O urea + 2ADP + 4Pi + AMP + 2H+

However, through linkage of the pathways the toll is not so bad. Some NADH is produced which regains about 2.5 ATP form respiration.

29

JA Negrn, Ph.D.

Glutamine transports NH3 in the bloodstream from peripheral tissues to the liver
Nucleotide degradation in other tissues produce NH3, which needs to be transported to the liver for processing. Glutamate accepts the NH3 by the action of glutamine synthetase.
30

JA Negrn, Ph.D.

15

5/10/2012

Amino Acid Degradation

Because of the structural diversity of the side chains of the twenty amino acids, their carbon skeletons do not all undergo identical degradation. Yet, they will form six major products all of which converge on the citric acid cycle.

31

JA Negrn, Ph.D.

Glucogenic and Ketogenic Amino Acids

Glucogenic amino acids: Degraded to pyruvate, or to one of the 4- or 5carbon intermediates of Krebs Cycle that are precursors for gluconeogenesis. Glucogenic amino acids are the major carbon source for gluconeogenesis when glucose levels are low. Ketogenic amino acids: Their carbon skeletons are degraded to acetyl-CoA or acetoacetate. Acetyl CoA, and its precursor acetoacetate, cannot yield net production of oxaloacetate, the precursor for the gluconeogenesis pathway. Carbon skeletons of ketogenic amino acids can be catabolized for energy in Krebs Cycle, or converted to ketone bodies or fatty acids. They cannot be converted to glucose.

32

JA Negrn, Ph.D.

16

5/10/2012

Glycogenic and Ketogenic Amino Acids

Glycogenic
Alanine, Arginine Asparagine, Aspartate Cysteine, Glutamate Glutamine, Glycine Histidine, Methionine Proline, Serine Threonine, Valine

Glycogenic and Ketogenic

Isoleucine Phenylalanine Tryptophan Tyrosine

Ketogenic
Leucine Lysine

Glycogenic: converted to glucose via pyruvate Ketogenic: converted to ketone bodies During fasting when FA are the major fuel FA cannot be converted to glucose therefore AA glucose & ketone bodies (especially for brain) -AA pyruvate liver glucose -keto AA + FA ketone bodies (acetoacetate & 3 hydroxybutyrate)
33

JA Negrn, Ph.D.

Glycogenic and Ketogenic Amino Acids

34

JA Negrn, Ph.D.

17

5/10/2012

Energy: Glucogenic Amino Acids

When the body's energy sources are low, it begins to degrade proteins for use as an alternative energy source. Amino acids can be classified as glucogenic or ketogenic. Glucogenic amino acids can be degraded to pyruvate or an intermediate in the Krebs Cycle. They are named glucogenic because they can produce glucose under conditions of low glucose (gluconeogenesis). , Amino acids form glucose through degradation to pyruvate or an intermediate in the Krebs Cycle.
JA Negrn, Ph.D.

Glucogenic Amino Acid: alanine, cysteine, glycine, serine, threonine, tryptophan, asparagine, aspartate, phenylalanine, tyrosine, isoleucine, methionine, threonine, valine, arginine, glutamate, glutamine, histidine, and proline
35

Energy: Ketogenic Amino Acids

Ketogenic amino acids can produce ketones when energy sources are low. Some of these amino acids are degraded directly to ketone bodies such as acetoacetate

Ketogenic Amino Acids: leucine, lysine, phenylalanine, tryptophan, and tyrosine

36

JA Negrn, Ph.D.

18

5/10/2012

One-carbon Transfer Reactions

Along with the wide variety of other reactions involved in amino acid degradation we need to consider one-carbon transfer reactions. Typically need cofactors that function as the carbon unit carriers for the enzyme.

37

JA Negrn, Ph.D.

One-carbon Transfer Reactions

Cofactors for one-carbon transfer reactions in amino acid degradation.

Tetrahydrofolate (H4 folate) Transfers carbon in intermediate oxidation states, sometimes methyl.
S-adenosylmethione (SAM or adoMet) Transfers carbon as methyl groups.
38

JA Negrn, Ph.D.

19

5/10/2012

39

40

20

5/10/2012

Trp, tryptophan
Most complex of all the amino acids. Four of its carbons yield acetoacetyl-CoA.

Some of the intermediates in Trp degradation.

Nicotinate, precursor of NAD and NADP. Serotonin, a neurotransmitterin vertebrates. Indoleacetate, growth factor in plants
41

JA Negrn, Ph.D.

Phe, phenylalanine
Breakdown products include acetoacetyl-CoA and fumarate.

After hydroxylation to Tyr is a precursor of the neurotransmitter, dopamine, and hormones epinephrine and norepinephrine.
Defects in enzymes of this pathway lead to several inheritable diseases.
42

JA Negrn, Ph.D.

21

5/10/2012

Phenylketonuria (PKU)
Caused by a genetic defect in phenylalanine hydroxylase, the first enzyme in the catabolic pathway for Phe. Results in elevated levels of Phe. Can cause mental retardation. Is easily detected with simple testing upon birth, retardation can then usually be avoided.
43

JA Negrn, Ph.D.

a-ketoglutarate
The carbon skeletons of five amino acids can enter the citric acid cycle as a-ketoglutarate. Pro, Glu, Gln, Arg, and His.

44

JA Negrn, Ph.D.

22

5/10/2012

Succinyl-CoA
The carbon skeletons of four amino acids can enter the citric acid cycle as succinyl-CoA.

Met, Ile, Thr, and Val

45

JA Negrn, Ph.D.

Oxaloacetate
The carbon skeletons of two amino acids can enter the citric acid cycle as
Asp and Asn

46

JA Negrn, Ph.D.

23

5/10/2012

Mostly in the liver

Most of the degradation of amino acids takes place in the liver, but three of the branched side chain amino acids are oxidized as fuel primarily in muscle, adipose, kidney and brain tissue. Leu, Ile, and Val

47

JA Negrn, Ph.D.

24