Removal of Nitrogen from Amino Acids

1 BIOSYNTHESIS OF UREA Removing the α-amino group is essential for producing energy from any amino acid, and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted as urea, with the carbon skeletons being metabolized. Urea biosynthesis occurs in four stages: (1) Transamination, (2) Oxidative deamination of glutamate, (3) Ammonia transport, and (4) Reactions of the urea cycle GENERAL STRATEGY  REMOVAL OF N FROM AMINO ACID BY transamination (generally first or second step of amino acid catabolic pathways) and collection of N in glutamate  Deamination of glutamate with release of + NH4 by glutamate dehydrogenase Collection of N in glutamine or alanine for delivery to liver glutamate dehydrogenase +  Removal of NH4 by : i. secretion; or ii. Conversion to urea or other less toxic form.

3 1. TRANSAMINATION 5 Transamination Reactions • Transfer of an amino group from an α -amino acid to an α-keto acid

• Transamination reactions generate GLUTAMATE or ASPARTATE • Nitrogen of most AA is concentrated in glutamate • Carried out in the cytosol and mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. TRANSAMINATION

α-ketoglutarate & glutamate are often involved in transamination reactions

One of the two pairs is almost invariably glutamate and its corresponding keto acid alpha ketoglutarate

All transaminases require pyridoxal phosphate PLP (derived from vitamin b6) as a coenzyme.

7 •Transamination: - Reversible (according to the body’s needs). - Requires pyridoxal phosphate as coenzyme. - Transfers an amino group from an amino acid to α-ketoglutarate. - Results in an α -keto acid and glutamate. * Note except in one reaction: oxaloacetate where it is the one that receives the amino group from glutamate and forms aspartate – why? Aspartate is the nitrogen source in the urea cycle. Transamination: the funneling of amino groups to glutamate The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate . The products are an α- keto acid (derived from the original amino acid) and glutamate. α- Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thus becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated , or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (formerly called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. *Note: These two amino acids lose their α-amino groups by deamination.] Substrate specificity of aminotransferases: Each aminotransferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always α-ketoglutarate. The two most important aminotrans•ferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), • These enzymes are found in the cytosol and mitochondria of cells- • Liver, kidney ,intestine and muscle.

• Alanine aminotransferase

• Aspartate aminotransferase Aspartate aminotransferase (AST): AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST transfers amino groups FROM glutamate TO oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle .[Note: The AST reaction is also reversible.] Glutamate + Oxaloacetate ↔ a-Ketoglutarate + Aspartate This reaction is an exception because glutamate transfers its amino group to oxaloacetate (instead of α -ketoglutarate) & produces aspartate (from OAA) which is the source of nitrogen, and α -ketoglutarate (from glutamate). Mechanism of action of aminotransferases: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. i.e 1- aminotransferases transfer amino group to pyridoxal-P forming pyridoxamine-P 2- pyridoxamine-P gives the amino group to a-keto acid , forming A.A. 3- pyridoxamine-P will return to its original aldehyde form (pyridoxal-P) by giving this amino group. Pyridoxal phosphate: NOTES The coenzyme (or prosthetic group) of all transaminases is pyridoxal phosphate, which is derived from pyridoxine (vitamin B6), and which is transiently converted into pyridoxamine phosphate during transamination. In the absence of substrate, the aldehyde group of pyridoxal phosphate forms a covalent Schiff base linkage (imine bond) with the amino group in the side- chain of a specific lysine residue in the active site of the enzyme. On further of substrate, and the α-amino group of the incoming amino acid displaces the amino group of the active site lysine and a new Schiff base linkage is formed with the amino acid substrate. The resulting amino acid-pyridoxal phosphate-Schiff base that is formed remains tightly bound to the enzyme through multiple noncovalent interactions. The amino acid is then hydrolyzed to form an pyridoxamine and α-keto acid Vitamine B6 family

Pyridoxine Pyridoxal Pyridoxamine

The substrates bind to the active centre to e-amino of lysine one at a time, and the function of the pyridoxal phosphate is to act as a temporary store of amino groups until the next substrate comes along. In the process the pyridoxal phosphate Pyridoxal phosphate is converted into pyridoxamine phosphate, and then back again. Pyridoxal phosphate as cofactor for aminotransferase NOTES

During degradation of amino acids (A A):

-A A approaches the active site of aminotransferase

-The amino group of A A displace the lysine amino group and form “Schiff base” linkage with pyridoxal phosphate.

- Hydrolysis of A A will give : α-keto acid and pyridoxamine

16 SCHIFF BASE NOTES • Schiff base is a functional group that contains a C=N double

bond with N attached to an Pyridoxal phosphate aryl/alkyl group bout not hydrogen. • (Biochemistry) the product of R CH COOH

the chemical association of an NH2 aldehyde with a primary amine H O • named after Hugo Schiff (1834- C O HO CH2O P OH 1915), German chemist OH

H3C N

Pyradoxamine phosphate Pyradoxamine ketoacid 1 - - a

COO

- +

- C - H O

1 R

2

NH

protein - Lys

Schiff base with substrate with base Schiff

protein - Lys

COO - C - NH H -

P - e pyridoxamin P - pyridoxal 

tion transamina of Phase .

acid acid o ket aminoacid  ■Diagnostic value of transamination :

Transaminases are normally intracellular enzymes. They are elevated in the blood when damage to the cells producing these enzymes occurs. * Increase level of both ALT & AST indicates possible damage to the liver cells. * Increase level of AST ALONE suggests damage to heart muscle , skeletal muscle or kidney. • ALT is more specific than AST for liver disease. • ALT is found more in the liver while AST is found everywhere. 2. OXIDATIVE DEAMINATION OF GLUTAMATE OXIDATIVE DEAMINATION OF GLUTAMATE. Glutamate dehydrogenase: the oxidative deamination of amino acids

In contrast to transamination reactions that transfer amino groups, oxidative deamination by glutamate dehydrogenase results in the liberation of the amino group as free ammonia (NH3) . These reactions occur primarily in the liver and kidney. They provide a) α-keto acids that can enter the central pathway of energy metabolism, and b) ammonia, which is a source of nitrogen in urea synthesis. DEAMINATION The amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate.

Glutamate is the only amino acids which rapidly undergoes rapid oxidative deamination .

. α- a.a. ◄ ► α- keto glutarate ◄ ► NH3 ► NAD(P)H+H GLUTAMATE TRANSAMINASE GLUTAMATE TRANSAMINATION DEHYDROGENASE NAD(P) OXIDATIVE DEAMINATION ► H2O α- keto acid ◄ ► Glutamic acid ◄ Glutamate dehydrogenase:

• This enzyme is the first committed step on the final common pathway for mammalian nitrogen excretion, leading eventually to urea. • about 75% of ingested protein nitrogen follows the glutamate route. Intracellular localization

Transamination  glutamate

In hepatocytes, cytosol glutamate is transported from the mitochondria cytosol into Glutamate glutamate NH3 Dehydregenase mitochondria, where it undergoes oxidative urea synthesis cytosol deamination catalyzed by L-glutamate Glu + NH3  Gln dehydrogenase

24 Glutamate dehydrogenase in mammals is almost entirely confined to the liver and kidney mitochondrial matrix space, where it accounts for a significant proportion of the total protein

It is the only enzyme that can use either NAD or NADP as the acceptor of reducing equivalents The process is therefore termed "oxidative deamination". It is the only common dehydrogenase which is non-specific for NAD or NADP, and this may be important for its overall regulation.

NAD- oxidative deamination ( loss of ammonia) NADP- reductive amination ( gain of ammonia) Glutamate dehydrogenase: NOTES As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination—a reaction catalyzed by glutamate dehydrogenase .Therefore, the sequential action of transamination (resulting in the collection of amino groups from most amino acids onto α-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating α- ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.In other words , this enzyme is found in many tissues, where it catalyzes the reversible oxidative deamination of the amino acid glutamate. It produces the citric acid cycle intermediate α-ketoglutarate, which serves as an entry point to the cycle for a group of glucogenic amino acids • In contrast to the transamination reactions which merely swoops amino groups from one compound to another

• GluDH catalyses a net loss of nitrogen from the amino acid pool i.e. liberation of amino group as free ammonia.

• the amino groups from many of the amino acids are collected in the liver in the form of the amino group of L-glutamate molecules.

• These amino groups must next be removed from glutamate to prepare them for excretion.