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Chart 51.1: nitrogen, in the form of ammonium ions or glutamate, is used for urea synthesis

Chart 51.1: nitrogen, in the form of ammonium ions or glutamate, is used for urea synthesis

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girls, the condition can vary from being undetectable to a severity equal

to that in boys. In this condition, carbamoyl phosphate accumulates

and  passes into the cytosol where it reacts with aspartate to form

­carbamoyl aspartate. This is metabolized to form orotate by the reactions described for pyrimidine synthesis in Chapter  55. The detection

of  ­

orotic  acid (­orotic aciduria) in urine is used to diagnose OTC

deficiency.



Creatine and creatinine



OTC deficiency and gene therapy



Purine nucleotide cycle



The main function of the ornithine cycle is to produce urea. However, as shown

in the chart, a small but significant quantity of arginine is diverted to form

creatine. This is phosphorylated by creatine kinase to produce creatine phosphate, which is the phosphagen used to generate ATP during short bursts of

intensive exercise. Approximately 2% of the body pool of creatine phosphate

spontaneously cyclizes each day and is excreted in the urine as creatinine.



There was considerable optimism that OTC deficiency would be a model

candidate for liver‐directed gene therapy. Unfortunately, a pilot study on 17

subjects with partial OTC deficiency using an adenoviral vector was very

disappointing. There was very little gene transfer and when subject 18 suffered lethal complications, the trial was stopped.



The purine nucleotide cycle described by Lowenstein, although present in

many types of tissues, is particularly active in muscle. During vigorous exercise in rats, the blood concentration of ammonium ions can increase five‐

fold. This ammonium is thought to be derived from aspartate via the purine

nucleotide cycle. This cycle is mentioned in Chapter 19.



alanine



tryptophan



lysine



Liver



α-KG



orotate

phenylalanine

dihydroorotate



α-KG



tyrosine



aspartate



cysteine



α-ketoglutarate



carbamoyl

aspartate



ornithine



glutamate

α-aminoadipate



proline



histidine



mitochondrion



serine



threonine



oxaloacetate



pyruvate



pyruvate



methyl

glyoxal



acetyl CoA



glutamate



asparagine

NH +

4



NH +

4



NH4+



aspartate



α-ketoacid



α-ketoglutarate



pyruvate

4-hydroxyphenylpyruvate



α-ketoacid



α-ketoadipate



3-hydroxypyruvate



methionine



+

NH4



+

NH4



+

NH4



alanine



glutamate



This pathway

operates in some

urea cycle disorders



serine



pyruvate



aminotransferase



orotic

aciduria



saccharopine



urea



alanine



see

Chapter 50



arginine



glutamate



aminotransferase



succinyl CoA succinyl CoA



glutamate



glutamate

α-ketoacid

(oxaloacetate)



glutamate

fatty acids



Cytosol



β-Oxidation

COO-



COO-



carnitine shuttle



C14



H3+NCH



glutamate



C12



acetate



arginine



H2C COO-



CH2



CH2 COO-



acetyl CoA



N-acetylglutamate

synthase



glutamate



C8



COO-



H2O

O



C6



CH3 C



O



H

N



NH



inactive



inactive CPS



+

HCO 3



glutamate



+

2ADP+Pi+3H



glutamine



NH4+



glutamate



+NH



3



ornithine

glycine



CH3

NH2

3



creatine



C

+NH



COOCH2



glycine

transamidinase



NH

C



ornithine



2



NH2



+NH

2



NH2



S-adenosylmethionine



methyl

transferase



guanidinoacetate



arginine



COO-



(CH2)3



+NH



COOfumarate



3



NH



OTC

deficiency



H3+NCH



C



HC



CH2



COOH +NCH



Urea cycle



H O

2



arginase



S-adenosyl

homocysteine



COOH3+NCH

(CH2)3

NH2



ornithine



Mitochondrion

(i) to intestines for fuel

(ii) to kidney for acid/base

regulation



2



CH2

N



CH



COO-



(CH2)3



NH2



CO2



ADP+Pi



glutamine synthetase



CH



COO-



3



carbamoyl

phosphate



NH4+

ATP



NH



COO-



argininosuccinate



ornithine

transcarbamoylase

(OTC)



Pi



Transdeamination route



α-ketoglutarate



+NH



COO-



CH2



C



citrulline



C O

+NH



active

carbamoyl phosphate

synthetase (CPS)

H O

2



NH



lyase



PO42-



H2O



COO-



(CH2)3

AMP+PPi



synthetase



citrulline



fumarase

(cytosolic)



H3+NCH



O



NH2



active



2 ATP



+

NADH+H



H O

2

NAD+



ATP



CPS



acetyl CoA + acetoacetate

succinyl CoA

succinyl CoA + acetyl CoA



glutamate

dehydrogenase



C



malate



COO-



aspartate



(CH2)3



H2C COO-



Transamination

route



COO-



H3+NCH



N-acetylglutamate

(NAG)

CPS



CH2



COO-



Pi



CH 2COO-



C4



H3+NCH



aspartate

transcarbamoylase



CH 2COOCH



CHOH



aspartate

aminotransferase (AST)



hydrolase



CoA



COO-



NAD+



malate dehydrogenase



oxaloacetate

α-ketoglutarate



C10



+

NADH+H



C O



O

C NH 2

NH 2



urea



+

NH4



Part 5  Amino acid metabolism



103



Metabolic channelling I: enzymes are organized to enable channelling

of metabolic intermediates

When I was a student it was rather assumed that the cell was ‘a bag of

enzymes’ and that their substrates were randomly moving throughout the

cytoplasm until a chance collision brought enzyme and substrate together,

enabling the reaction to proceed. It was imagined that the product of this

reaction would diffuse through the aqueous environment until a chance

encounter with the next enzyme and substrate occurred to form the next

product, and so on through to the end of the metabolic pathway. However,

this simplistic idea is very inefficient and P. A. Srere introduced the hypothesis of metabolic channelling. This concept proposes that the products of

an enzyme reaction are passed directly from the enzyme to the next enzyme

in the metabolic sequence. This was defined by Srere as follows:

‘Metabolic channelling of an intermediate can be defined as the passage of a

common intermediate between two enzymes. The intermediate is localised

and is out of equilibrium with the bulk solution.’



Experimental evidence supporting ‘metabolic channelling’

Co‐precipitation of enzymes



One of several experimental approaches that provides compelling evidence

in support of metabolic channelling is provided by the tendency of enzymes

that are sequential in a metabolic pathway to associate and co‐precipitate

when studied under certain conditions. Although enzymes when studied in

dilute solution in vitro are free to diffuse in search of their substrates, this is

not the case in vivo. For example, the proportion of soluble protein in the

mitochondrial matrix is approximately 500 mg/ml of water. This water is

approximately 50% water of hydration leaving just 50% free. These

­conditions can be simulated in vitro by adding to an enzyme preparation a

volume‐excluder, such as polyethyleneglycol, that removes water causing

the enzymes to crowd together. This results in enzymes that are next to each

other in a metabolic sequence to associate and co‐precipitate. For example,

the mitochondrial enzyme citrate synthase has been shown to bind to and

co‐precipitate with pyruvate carboxylase, the pyruvate carrier, pyruvate

dehydrogenase and the tricarboxylate transporter. As shown in Diagram

52.1, these enzymes and carrier proteins are sequential in the pathways for

pyruvate metabolism in mitochondria. Moreover, the binding is specific; for

example, citrate synthase binds to the mitochondrial isoform of malate

dehydrogenase but not to the cytosolic isoform of this enzyme.



­ roduct D. If the substrates and their enzymes are free to diffuse in solution

p

(i.e. if there is no channelling of the metabolites), then at steady state the

specific activity of B, C and D will be the same as A. If, as shown in Diagram

52.2b, a 200‐fold excess of non‐radioactive C is added, then in the absence

of channelling the specific radioactivity of C and product D will be diluted

200‐fold, i.e. to 0.5% of the original value.

However, as represented metaphorically in Diagram 52.2c, if the metabolites and their enzymes are prevented from freely diffusing in the surrounding solution (i.e. the intermediates are passed from enzyme to enzyme and

metabolite channelling is occurring), then the addition of a 200‐fold excess

of non‐radioactive C will not dilute the specific radioactivity of product D.

Instead, D is formed from the channelled radioactive C rather than from the

pool of non‐radioactive C in the bulk solution.



Metabolic channelling in the urea cycle

Experiments using radioisotope dilution studies suggest that metabolic

channelling occurs in the urea cycle, albeit incompletely (Diagram 52.3).

An experiment used α‐toxin to make pores in the plasma membrane of

hepatocytes. The hepatocytes were incubated in a physiological medium

with 14C‐labelled HCO3–, aspartate and ammonium chloride as carbon

and nitrogen sources plus other essential compounds. The 14C label

appeared in urea as would be anticipated. When a 200‐fold excess of non‐

radioactive arginine was added, there was no decrease in the specific radioactivity of the urea formed. This suggests that metabolic channelling



Diagram 52.2a Metabolism of radioactive substrate A to product D.



A



B



C



Diagram 52.2b No metabolic channelling.



C



Isotope dilution studies



pyruvate



intermembrane

space



citrate



A

Diagram 52.2c Metabolic

channelling present.



pyruvate

carrier



inner

mitochondrial

membrane



pyruvate

pyruvate

carboxylase



matrix



oxaloacetate



malate



malate

dehydrogenase



oxaloacetate



C



D



C



pyruvate

dehydrogenase



acetyl CoA



A

citrate

synthase



tricarboxylate

carrier



citrate



Diagram 52.1  Enzymes associating with citrate synthase. A schematic representation of how the enzymes and carrier

proteins involved in the reactions adjacent to citrate synthase might be organized to allow metabolic channelling.



104



B

2

no 00n- fol

ra d

di ex

oa ce

ct ss

iv o

e f

C



Further evidence for metabolic channelling is provided by radioisotope

dilution studies. Diagram 52.2a represents a metabolic pathway in which a

radiolabelled substrate A is metabolized via intermediates B and C to



D



2

no 00n- fol

ra d

di ex

oa ce

ct ss

iv o

e f

C



52



Metabolic intermediates are channelled from enzyme

to enzyme



B



C



D



Diagram 52.2  Experimental approach to demonstrate metabolic channelling by

radioisotope dilution. (a) This represents a pathway from substrate A, which is

metabolized via intermediates B and C to product D. If A is radioactive, then the specific

radioactivity of intermediates B and C and product D will all be the same. (b) If a 200‐fold

excess of non‐radioactive C is added, then provided substrate channelling does not occur,

radioactive C will equilibrate with non‐radioactive C and the specific radioactivity of

product D will be diluted 200‐fold. (c) If the experiment in (b) is repeated but metabolic

channelling does occur, then the 200‐fold excess of non‐radioactive C will not equilibrate

with radioactive C and the specific radioactivity of D will be the same as A.



Metabolism at a Glance, Fourth Edition. J. G. Salway. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.



Experiment: To determine if

arginine is channelled between

lyase and arginase



Experiment: To determine if

citrulline is channelled between

ornithine transcarbamoylase and

arginosuccinate synthetase



Method: Add a 200-fold excess of

non-radioactive arginine

Result: NO reduction of specific

radioactivity of urea

no 2

n- 00

ra -fo

di ld

oa e

ct xc

iv e

e ss

ar o

gi f

ni

ne



Conclusion: Non-radioactive

arginine does NOT mix with

radioactive arginine suggesting

metabolic channelling occurs

between lyase and arginase



Result: Partial reduction of specific

radioactivity of citrulline and urea

Conclusion: Partial mixing of added

citrulline occurs when it crosses the

intermembrane space. This suggests that

partial channelling occurs at this stage



arginine



no 2

n- 00

ra -f

di ol

oa d

ct exc

iv e

e ss

ci o

tr f

ul

lin

e



Method: Add a 200-fold excess of

non-radioactive citrulline



α toxin



Plasma membrane



citrulline



α toxin



arginine



urea



Cytosol



arginine

ornithine



Mitochondrial

outer membrane



arginase



lyase



aspartate



argininosuccinate

synthetase



porin



Intermembrane

space



Mitochondrial

innner

membrane



argininosuccinate



ornithine



carbamoyl

phosphate

synthetase



citrulline



urea cycle



citrulline



transporter



non-radioactive citrulline can

mix with radioactive citrulline

as it diffuses across the

intermembrane space



transporter

ornithine

transcarbamoylase



carbamoyl

phosphate



citrulline

Diagram 52.3  Partial metabolic

channelling in the urea cycle.

Channelling is interrupted when

ornithine and citrulline diffuse across

the intermembrane space. During

this stage of the journey, the

molecules are free to equilibrate with

other molecules in the intermembrane space and so metabolic

channelling does not occur.



Matrix

HCO3–

bicarbonate

(radioactive)

NH4+

ammonium



occurs between the lyase and arginase enzymes. However, when a 200‐fold

excess of non‐radioactive citrulline was added, the specific radioactivity

of the urea formed was reduced. This is because citrulline is formed by

enzymes on the mitochondrial inner membrane and must diffuse across

the intermembrane space to argininosuccinate synthetase, which is

located on the outer side of the outer mitochondrial membrane. While 14C

citrulline is diffusing across the intermembrane space metabolic channelling



is not occurring and the radiolabelled citrulline is diluted with the added

non‐radioactive citrulline.



Reference



Cohen N.S., Cheung C.W., Raijman L. (1996) The urea cycle. In: Channelling

in Intermediary Metabolism (L. Agius & H.S.A. Sherratt, eds), pp. 183–99.

Portland Press, London and Miami.



Part 6  Metabolic channelling



105



Metabolic channelling II: fatty acid synthase



53



4 Enoyl ACP reductase (ER) (also known simply as enoyl reductase).

5 Thioesterase (TE). Once the fatty acyl chain is complete (palmitate is

formed), it is thiolytically cleaved from ACP by thioesterase.

6 Malonyl‐acetyl CoA‐ACP transacylase (MAT) (also known as malonyl/

acetytransferase). This transfers the malonyl group of malonyl CoA to

ACP, forming malonyl ACP. It also transfers the acetyl group of acetyl

CoA to ACP, forming acetyl ACP. This acetyl group provides the ω and

ω‐1 carbon atoms of the fatty acid chain with all subsequent carbon

atoms being provided by malonyl CoA.

In addition, there are two proteins:

1 Acyl carrier protein (ACP). ACP is a relatively small protein of 54 kD.

The prosthetic group phosphopantetheine (Diagram 53.3a) is attached

to serine‐2151 (Diagram 53.3b). This long prosthetic group carries the

acyl groups sequentially from enzyme to enzyme as they grow, in the

manner of a robotic arm on an assembly line which would rival a modern

motor car factory.

2 Core protein. The core protein of each monomer is a component that

stabilizes the structure of the dimer and is without enzymic activity.



The de novo biosynthesis of palmitate from its precursor acetyl CoA involves

the formation of 34 intermediate metabolites, which takes ­metabolic channelling

to an extraordinary level of sophistication. It is repre­sented in its familiar format

in Chart 53.1 and as a cartoon in Diagram 53.4.



Fatty acid synthase complex

In animals, fatty acid synthase consists of two polypeptide chains. The two

subunits are identical and are organized in a head‐to‐toe configuration

(Diagrams 53.1 and 53.2). The component enzymes of the fatty acid synthase complex (Diagram 53.2) are:

1 β‐Ketoacyl ACP synthase (KS) (also known as condensing enzyme or

3‐oxoacyl synthase). The sulphydryl group of cysteine 161 has a vital

function in the ‘condensation reaction’. This is the process of chain elongation that occurs when malonyl acyl carrier protein (ACP) condenses

with acyl ACP (or the initial acetyl ACP).

2 β‐Ketoacyl ACP reductase (KR) (also known as 3‐oxoacyl reductase).

3 β‐Hydroxyacyl ACP dehydratase (HD) (also known as 3‐hydroxyacyl

hydratase).



Subunit 1



Subunit 2

Diagram 53.1  In animals, fatty acid synthase is a dimer of two subunits

that associate to form a complex with two holes.

O



O

H 3C



C



acetyl CoA

malonyl-acetyl CoA-ACP

transacylase (MAT)



NADPH+H+



HS-ACP



H



H3C C



C



NADPH+H+



SACP



H3C CH2



hexanoyl ACP



H2O

thioesterase

(TE)



CH2 C

acyl ACP



SACP



CO2



HS–KS



CO2



C10



C8



C6



CO2



CO2



C12

CO2



C14

CO2



C16



acyl

carrier

protein

(ACP)



CO2



condensation



condensation



O



-O C



CH2 C



HS-ACP

SACP



malonyl ACP

CoASH



CoASH



—SH of acyl carrier protein (ACP)



CoASH



acyl-KS



O



malonyl-acetyl CoA-ACP

transacylase (MAT)



O

O

-O C CH C

2

malonyl CoA



SCoA



Chart 53.1  Reactions of the fatty acid synthase complex.



TE

Core

Protein



HD

β-hydroxyacyl

ACP

dehydratase



ER



ACP



KR



Subunit 2



Diagram 53.2  The protein components of each monomer are

organized in a head‐to‐toe manner. For example, in the diagram

thioesterase (TE) of subunit 1 is on the left, while TE of subunit 2 is on

the right. This arrangement allows cooperation between the subunits;

that is that TE, ACP, KR and ER of subunit 1 collaborate with HD, MAT

and KS of subunit 2 (and vice versa for the similar enzymes on the right

of the diagram).



O



translocation



β-ketoacyl-ACP synthase (KS)

(condensing enzyme)



KS



HS



palmitoyl ACP



O



MAT



HS-cysteine



MAT



enoyl ACP

reductase (ER)



NADP+



HD



Core

Protein



SH



malonyl-acetyl

CoA-ACP

transacylase



H

enoyl ACP



acetyl—KS



106



C



ER



β-ketoacyl ACP

KS cysteine-SH

synthase

(condensing

enzyme)



O



H

HS-ACP



KR



TE



β-hydroxyacyl ACP

dehydratase (DH)



H2O



enoyl

ACP

reductase



β-ketoacyl

ACP

reductase



thioesterase



CH2 C SACP



OH

D-3-hydroxybutyryl ACP



SACP



cysteine-SH of KS

(condensing enzyme)



C4



ACP



O



H3C C



acetyl ACP



acetoacetyl ACP



Fatty acid synthesis



acyl carrier

protein



β-ketoacyl ACP

reductase (KR)



NADP+



O

C



Subunit 1



acetoacetyl ACP

C4



CoASH



H3C



O



H3C C CH2 C SACP



SCoA



CoASH



CoASH



CoASH



CoASH



CH3(CH2)14C O-



palmitate



CH2 O

(a)



malonyl-acetyl

CoA-ACP

transacylase

(MAT)



ACP



malonyl CoA



O–

P CH2



CH3

C



H

C



O



CH3



OH O



CH2 O



C



O–

P CH2

O



H

N



CH2 CH2 C



H

N



CH2 CH2 SH



O



CH3

C

CH3



H

C

C

OH O



H

N



CH2 CH2 C

O



H

N



CH2 CH2



SH



(b)



Diagram 53.3  (a) Phosphopantetheine. (b) Phosphopantetheine is

attached to acyl carrier protein (ACP) to form a long prosthetic group.



Metabolism at a Glance, Fourth Edition. J. G. Salway. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.



1



The acetyl group of

acetyl CoA combines

with ACP in a reaction

catalysed by malonylacetylCoA-ACP

transacylase (MAT)



O

H3C



SCoA



C



acetyl CoA

S



O



H



malonyl-acetyl

CoA-ACP

transacylase

(MAT)



ACP

CoASH



O

H3C CH2



acetoacetyl ACP (C4)

NADPH+H



O



CH2



CH2



CH2 C

hexanoyl ACP (C6)



ACP



S



palmitoyl ACP (C16)



ACP



β-ketoacyl ACP

reductase

(KR)



+



NADP+

OH



H3C C S



Fatty acid synthesis



O



H3C C CH2 C S



H2O



thioesterase

(TE)



O



H



S



H3C CH CH2 C S



D-3-hydroxybutyryl ACP ACP



ACP



acetyl ACP

H2O



H S-cysteine KS



ACP



β-hydroxyacyl

ACP dehydratase

(DH)



β-ketoacyl-ACP synthase (KS)

H



S



O



H

H3C C



ACP



C



C



S



H

enoyl ACP

O



enoyl ACP

reductase

(ER)



NADPH+H+



S-cysteine KS



H3 C C



NADP+



acetyl KS



ACP



O

H3C CH2



S



CH2 C



C4 acyl ACP (butryl ACP) ACP



O



O



O



H3C C CH2 C S



acetoacetyl ACP (C4)



H3C CH2



CH2



CH2



CH2 C



S



hexanoyl ACP (C6)



ACP



C8



ACP



C10



C12



C14



C16



H S-cysteine KS



Translocation



Transfer of acyl group from

phosphopantetheine of ACP

to cysteine-SH of KS.



Condensation



Condensation



Chain-elongation catalysed

by KS. Acetyl group on

cysteine-SH of KS reacts

with malonyl group on the

phosphopantetheine-SH

of ACP.



CO2



Chain-elongation

catalysed by KS. Acyl

group on cysteine-SH of

KS reacts with malonyl

group on the

phosphopantetheine-SH

of ACP.



CO2



O

H3C CH2



CH2 C



CO2



S-cysteine KS



CO2



CO2



CO2



CO2



2



The malonyl group of malonyl

CoA combines with -SH of

the phosphopantetheine

prosthetic group of ACP

in a reaction catalysed by

malonyl-acetylCoA-ACP

transacylase (MAT)



O

O

-O C CH C

2

malonyl ACP



CoASH



ACP



malonyl-acetyl

CoA-ACP

transacylase

(MAT)



SH

ACP



S



O

O

-O C CH C

2

malonyl CoA



SCoA



Pi+ADP



O

O

-O C CH C

2

malonyl ACP



CoASH



H



ACP



O

O

-O C CH C

2

malonyl CoA



SCoA



CH3C



SCoA



acetyl CoA



O

O

-O C CH C

2

malonyl CoA



acetyl CoA

carboxylase

ATP



ATP

ATP

ATP

ATP

ATP



HCO3-



HCO3–

HCO3–

HCO3–

HCO3–

HCO3–



O



O



CoASH



CoASH



CoASH



CoASH



CoASH



SCoA



Pi+ADP

Pi+ADP

Pi+ADP

Pi+ADP

Pi+ADP



acetyl CoA

carboxylase

ATP



ACP



malonyl-acetyl CoA-ACP

transacylase (MAT)



Pi+ADP



HCO3-



S



S



CH3C



acetyl CoA

carboxylase



O

CH2



CH2



CH2



CH2



CH2



CH2



CH2



C



O–



O

SCoA



acetyl CoA



CH3C



SCoA



acetyl CoA



H3C



CH2



CH2



CH2



CH2



CH2



CH2



CH2



palmitate (C16)



Diagram 53.4  Reactions of the fatty acid synthase complex (cartoon version).



Part 6  Metabolic channelling



107



Amino acid metabolism, folate metabolism and the ‘1‐carbon pool’ I:

purine biosynthesis



54



Chart 54.1  (opposite)  Purine

biosynthesis. For an explanation of

low‐dose methotrexate functioning as

an antirheumatic drug, see

Chapter 55.



Glutamine plays a very important role in nucleotide metabolism. It

donates the nitrogen atoms that form N‐9 and N‐3 of the purine ring. It also

participates in the amination of xanthine monophosphate (XMP) to form

guanosine monophosphate (GMP) (Chart 54.2).



The ‘1‐carbon pool’

This term describes the 1‐carbon residues associated with S‐adenosylmethionine (SAM) and folate that are available for metabolic reactions.

S‐adenosylmethionine (SAM). SAM, which is formed from methionine, is

the major donor of methyl groups for biosynthetic reactions. It can, for

example, methylate noradrenaline to form adrenaline, as shown in

Chart 49.1. Other important reactions involving SAM include the methylation of phosphatidylethanolamine to phosphatidylcholine, and the formation of creatine.

Folate ‘1‐carbon’ units. The vitamin folate is reduced in two stages by dihydrofolate reductase to produce the active form, tetrahydrofolate (THF)

(Chart  54.1). THF is a versatile carrier of 1‐carbon units in the following

oxidation states: formyl, methenyl, methylene and methyl. These THF compounds, which are interconvertible, together with SAM, comprise what is

known as the ‘1‐carbon pool’.



Biosynthesis of purines

Purine nucleotides can be synthesized de novo. They can also be reclaimed

from existing nucleosides by the so‐called ‘salvage pathway’ (see Chapter 55).

The de novo pathway needs ‘1‐carbon’ units from the folate pool, and several

amino acids as detailed below.



De novo pathway for purine biosynthesis



The pathway starts with ribose 5‐phosphate formed by the pentose phosphate

pathway (Chart 54.1). This is activated to form phosphoribosyl pyrophosphate

(PRPP). A total of 11 reactions are needed to form IMP (or inosinic acid),

which is the precursor of the adenine‐ and guanine‐containing nucleotides. The

important roles of glutamine and aspartate as amino donors are emphasized.

A total of 3 glutamine molecules and 1 aspartate molecule are needed for the

synthesis of GMP. Similarly, a total of 2 ­glutamine and 2 aspartate molecules

are needed for AMP synthesis. A m

­ olecule of glycine is needed in each case.

The de novo pathway is controlled by feedback inhibition of PRPP amidotransferase by AMP and GMP. In primary gout this feedback control is

impaired, causing increased production of purines resulting in the increased

formation of their sparingly soluble excretory product, uric acid.



Amino acids and the ‘1‐carbon pool’

Serine is converted to glycine, in a reaction catalysed by serine hydroxymethyl transferase, with the transfer of a methyl group to THF forming

N5,N10‐methylene THF. This reaction is particularly important in the

­thymidylate synthase reaction described in Chapter 55. Oxidation of glycine

in mitochondria by the glycine cleavage enzyme also produces N5,N10‐­

methylene THF (see Chapter 46).

Tryptophan is oxidized to N‐formylkynurenine, which, in the presence of

formamidase, yields kynurenine and the toxic product formate. THF

accepts the formate, producing N10‐formyl THF.

Methionine, as mentioned above, is the precursor of SAM, which,

­following transfer of the methyl group to an acceptor, e.g. noradrenaline,

forms ­homocysteine. Methionine can be regenerated from homocysteine by

­methylation using N5‐methyl THF in a salvage pathway. NB: This reaction,

catalysed by homocysteine methyltransferase, requires vitamin B12, and deficiency can lead to folate being caught in the ‘methyl‐folate’ trap (see below).



Vitamin B12 and the ‘methyl‐folate trap’



Vitamin B12, or more precisely its methyl cobalamin derivative, is an essential

coenzyme for the transfer of methyl groups in the methionine salvage pathway

(Chart 54.1). Accordingly, in B12 deficiency, THF cannot be released and remains

trapped as N5‐methyl THF. Eventually, all the body’s folate becomes trapped in

the N5‐methyl THF form, and so folate deficiency develops secondary to B12 deficiency. Because blood cells turn over rapidly, they need nucleotides for nucleic

acid synthesis and are vulnerable to folate deficiency, which causes megaloblastic

anaemia. Another effect of folate/B12 deficiency is increased plasma concentration of homocysteine, which is associated with cardiovascular disease.

The methyl‐folate trap hypothesis explains the observation that, although

the haematological symptoms of B12 deficiency respond to folate treatment,

the neurological degeneration progresses. Remember that the other enzyme

for which B12 is a coenzyme is methylmalonyl CoA mutase (see Chapters 45

and 46). Accumulation of methylmalonyl CoA may interfere with the

­biosynthesis of lipids needed for the myelin sheath.



Amino acid metabolism and purine synthesis



Chart 54.2  Conversion of IMP to

ATP, the purine nucleotide cycle.

IMP reacts with aspartate in the

presence of GTP to form adenylosuccinate, which is cleaved to form

fumarate and AMP. The AMP can be

phosphorylated to ADP, which

undergoes oxidative phosphorylation

to form ATP. The purine nucleotide

cycle has an anapleurotic role in

Krebs cycle.

Conversion of IMP to GTP. IMP is

oxidized to xanthine monophosphate

(XMP), which is aminated to form

GMP, which is phosphorylated to

form GDP. GDP is phosphorylated by

ATP in a reaction catalysed by

nucleoside diphosphate kinase.

Alternatively, when Krebs cycle is

active, GTP is formed from GDP by

succinyl CoA synthetase (see

Chapter 19).

Formation of dATP (deoxyadenosine triphosphate) and dGTP

(deoxyguanosine triphosphate). The

deoxy‐ribonucleotides dATP and

dGTP are formed by first reducing

ADP and GDP to dADP and dGDP

in the presence of ribonucleotide

reductase. These are subsequently

phosphorylated to form dATP and

dGTP, which can be used for the

synthesis of DNA.



108



Glycine contributes the C‐4, C‐5 and N‐7 atoms to the purine ring in a reaction catalysed by glycinamide ribonucleotide (GAR) synthetase (Chart 54.1).

Aspartate is an important donor of nitrogen atoms during purine biosynthesis, contributing the N‐1 atom to the purine ring. Aspartate also donates

the –NH2 group in the adenylosuccinate synthetase reaction of the pathway

that forms AMP from inosine monophosphate (IMP) (Chart 54.2).



Mitochondrion

Inner membrane

ADP



nucleoside diphosphate kinase



4H+



ADP



ATP



NADH+H+

NAD+



I



translocase



1

2O



2



2H+

2H+



4H+

III



Q



H2O



IV



ADP HPO42- H+



ATP



F1



ATP



ADP3-



FO



translocase



C



ATP



ATP



ADP



ATP



ADP



3H+



Outer membrane



GTP



IMP



GDP

ADP



GDP



mercaptopurine



glutamine

GMP kinase



NADH+H+ NAD+



AMP+PPi ATP



ATP



GMP



XMP



synthetase



mercaptopurine



aspartate

α-ketoglutarate

aspartate

aminotransferase



GTP



oxaloacetate



RNA

polymerase



Cytosol



FAD



thioredoxin



(SH)2



thioredoxin

reductase



NADP+



FADH2



thioredoxin



adenylosuccinase



AMP



purine

nucleotide

cycle



AMP kinase



malate dehydrogenase



ATP

H2O



fumarase

RNA

polymerase



malate



ADP



RNA



2H+



2H+



S



DNA



S

ADP



DNA

polymerase



ADP



fumarate



NAD+



GDP



ADP



ribonucleotide

reductase



RNA

DNA



ATP



adenylosuccinate



NADH+H+



glutamate



GTP

NADPH+H+



GDP+Pi



synthetase

dehydrogenase



H2O



glutamate



ATP



H2O



dGTP



H2O



ATP



dGDP



Metabolism at a Glance, Fourth Edition. J. G. Salway. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.



H2O



dADP



ATP



ADP



DNA

polymerase



dATP



THF



THF

COOH3+NCH



(vitamin B12)



N 5-methyl THF



CH2



H3+NCH

CH2



CH3

N+ CH3



CH3



SH



homocysteine



CH2



CH3 N



COO-



–CH 3

yl

meth



CH3



methyl group

transferred to

acceptor

e.g. noradrenaline



CH2



betaine



COO-



dimethylglycine



methionine salvage

pathway



methyl transferase

S-adenosylhomocysteine

H O

2

adenosine



serine



Homocysteine: a risk-factor for cardiovascular disease.

Folate/B12 deficiency, and several inborn errors of metabolism, cause

increased plasma concentrations of homocysteine. This is associated

with coronary atherosclerosis, peripheral vascular disease,

cerebrovascular disease and thrombosis. It is thought that

homocysteine reacts with amino groups on LDLs. These then clump

together, are engulfed by macrophages and deposited as atheroma.

Elevated plasma concentrations of homocysteine can be decreased by

treatment with folic acid, vitamin B12, and betaine which enhance

metabolism of homocysteine to methionine. Alternatively, vitamin B6 may

enhance catabolism of homocysteine by cystathionine synthase.

NAD+



COOHCOH



2+



Mg



cystathionine

synthase

Vit B6



O



2



deaminase



to

mitochondrion



N



2

HN 3



Cytosol



N



1



H



8



7

6



5



4



9



N



O



N

5



8



1



NH



C



H



adenosine



7



N



OH



H 2N



aminoimidazolecarboxamide

ribonucleoside

(AICARiboside)



C



8



4



5



formate



2-



H



IMP



extracellular

fluid



9



N



H



H



OH



OH



8



4



5



aminoimidazole

-carboxamide

ribonucleotide

(AICAR)



histidine



H3+N



CH COOglutamate



histidinase



NH4+ ATP



urocanate

H2O



hydratase



H2N



glutamate

formiminotransferase

imidazoline

propionase



THF



FIGLU

(N-formiminoglutamate)



4-imidazolone5-propionate



H2O



2

HN 3



N



N



2

HN 3



1



4



O



O



)



H



cyclodeaminase



8



4



5



N

HN C

H



7

6



5



COO-



)



On



HC



CH (CH2)2 C



N



O



NH



C



(



H COON



O



ATP

ADP+Pi



O

N



C



1



6



NH



7



5C



8 CH



3 4C



9



H2N



)



On



N



H

H



7

6



H



H



H



OH



OH



adenylosuccinase

(ASase)



ASase

deficiency



N



C



1



6



7



5C



8 CH

9



N



9



8



4



)



(



H COO-



O



CH (CH2)2 C



N



On



5



6



O CH



N



H C



O

C



10



(



H COON



O



CH (CH2)2 C



H



1



4



O



N

8



5



8 CH

9



N formaminoimidazolecarboxamide

ribonucleotide

H

(FAICAR)

OH

H



IMP

cyclohydrolase



H2O



On



O

N



C

6



HN 1

HC



2

3



5C

4C



N



2-



O3POCH2



H

7

6



O

H

OH



)



7



4C



O3POCH2



H

N



methotrexate

Low-dose

methotrexate

used to treat

rheumatoid

arthritis



N



NH

2-



N H CH2



AICAR

transformylase



5C



3



N5, N10-methylene THF



9



OH



C



H2N



H

H



OH



aminoimidazole

-carboxamide

ribonucleotide

(AICAR)



O

1



2



7

6



H



THF



)



methylene THF

reductase deficiency



2

HN 3



N5-methyl THF



C



10



H



H2N

On



O



N



H



H



10



N , N -methenyl THF



+



C



N



1



N

O



O3POCH2



N10-formyl THF

5



succinylaminoimidazole

carboxamide

ribonucleotide

(SAICAR)



O



O3POCH2



H2N



H



NADP+



CH (CH2)2 C



OH



O



N5,N10-methenylTHF reductase



NADPH+H+



N5-formimino THF



CH2

10



(



OH



fumarate



O



H



H



SCAAIR

synthetase



H



N5,N10-methylene

THF reductase



H

9



CH (CH2)2 C



H COO-



N H CH2



O

H



glutamine

8



O



N carboxyaminoimidazole

ribonucleotide (CAIR)



H



aspartate



N10-formyl THF

C



8 CH

9



O



O3POCH2



H



O



7



5C



3 4C



2-



9



N



6



2-



aminoimidazole

ribonucleotide (AIR)



AIR

carboxylase



H2N



H COON



H

OH



C



-O



On



H



H

OH



O



COO-



N



1



O



NH



(



O



O3POCH2



H

H2N



ADP+Pi



glutamine synthetase



NH4+



8 CH

9



N



3 4C



2

HN 3



CH2



N



CO2



CH (CH2)2 C



C



H

H



formylglycinamidine

ribonucleotide (FGAM)



7



H2N



N5-formimino-THF



CH2COO-



3



H

OH



3 4C



2-



NH4+



H



H

OH



i



H2O



H2N

CH2 CH

+NH



7

6



O



NH



O



5C



H COON



O



H 10 N

O C

H



NADP+



NH



9



N H CH2



NADPH+H+



N



H



8 CH



9



H



THF

(tetrahydrofolate)



H

7

6



N



1



N5,N10-methenyl-THF

cyclohydrolase



H



C



4



ADP+P



H



O



AICAR accumulates

when low-dose

methotrexate is used

to treat rheumatoid

arthritis



COO-



On



N10-formyl-THF

synthetase



N



2

HN 3



8 CH



O



O3POCH2



)



CH2

ATP



H2N



N



H 2N



(



10



7



5C



3 4C



AICAR

inhibits

AMP

deaminase



CH (CH2)2 C



H



ADP+Pi



6



C



N H CH2

H

NH



O

C



O



NH



N



1



O



OO



7



H2 C 5



AIR

synthetase



methotrexate

trimethoprim

aminopterin



N



2

HN 3



phosphatase



1



N



O



H



H



OH



CH2

10



9



N



H



H2 N



COO-



9



NADP+



8 CH



H2O



AMP



(



H



DHF

(dihydrofolate)



H



NADPH+H+



H



Pi



H



dihydrofolate

reductase



N



O



HO



i



H



7

6



azaserine



ADP+P



H

N



H2N



N



O



3 4C



H O

2

ATP



O3POCH2



methotrexate

trimethoprim

aminopterin



HN 3 4



2



H 2N



H

OH



glutamate



2-



H2N



formylglycinamide

ribonucleotide (FGAR)



H



H

OH



FGAM

synthetase



H



glycine



inosine



NH4+



H



O



9



NH



O



O3POCH2



8 CH



ATP



O



dihydrofolate

reductase



O



extracellular adenosine

is a potent

anti-inflammatory

mediator



2-



H



CH2



NADPH+H+



pyruvate



adenosine



COO-



folate



NADP+



5C



7



C4



HN



CH3



ecto-5´nucleotidase



H

N



3



COO-



6



H

OH



O



to

mitochondrion



H3+NCH2



1



H

OH



H2C 5



O



serine

hydroxymethyl

transferase



H2 N



9



NH

glycinamide

H

ribonucleotide (GAR)



O



THF



+

4



10



AICARiboside

inhibits

adenosine

deaminase



C4



reductase



C



7



GAR

transformylase



NH



H2N



C



i



CH2NH2



5



glutamine



serine



C O



ADP+P



O3POCH2



N10-formyl THF



2-aminomuconate



NAD+



β 5-phosphoribosylamine



ATP



H



NADH+H+



CH2OH



pyruvate

kinase

Mg2+ K+



H2O



2-



dehydrogenase



+



COO-



COO-



H

OH



NAD+

NADH+H



α-ketobutyrate



H



H

OH



O



2-aminomuconate

semialdehyde



2-



N5,N10-methylene

THF



NH2



GAR

synthetase



phosphatase



THF



9



O



glycine



picolinate

decarboxylase

CO

2



homoserine



H3+NCH



CH2

phosphoenolpyruvate



Pi



3,4-dioxygenase



i



COO-



nucleoside

transporter



H



2-amino-3-carboxymuconate

semialdehyde



cystathionase



3-phospho

serine



COPO32-



gout



O3POCH2



3-hydroxyanthranilate



α-ketoglutarate



P



AMP, GMP, IMP

(feedback inhibition)



alanine



H O

2



+

NH

4



H O

2



glutamate

PP

i



kynureninase



O-



P O P O



O

O

5-phosphoribosyl

pyrophosphate (PRPP)



2-



H O

2



O-



O



PRPP

amidotransferase



H O

2



3-hydroxykynurenine



glutamate



H O

2



enolase

2+

Mg



NH4+



3-monooxygenase



+

NADP



3-phospho

hydroxypyruvate



CH2OH

2-phosphoglycerate



ATP



O2

+

NADPH+H



H

OH



glutamine



kynurenine



formate



H



O

H

OH



6-mercaptopurine

azaserine

6-diazo-5-oxoL-norleucine (DON)



α-ketoadipate



3-phosphoserine

α-ketoglutarate

aminotransferase



HCOPO32-



ADP



H



+

NADH+H



COO-



H O

2



C



O3POCH2

H



formamidase



-OOC(CH )

2 3



dehydrogenase



CH2OPO323-phosphoglycerate



phosphoglycerate

mutase



adenosyl

homocysteinase



O



H2O



cystathionine

cysteine



2-



N-formylkynurenine

O-



homocysteine

H O

2



glycolysis



adenosyl transferase



P +PP

i

i



S-adenosylmethionine (SAM)



OH



AMP



2,3-dioxygenase



H O

2



ATP



OH

ATP



O



2



OH



H



ribose phosphate

pyrophosphokinase



tryptophan



methionine



SAM



H



H



ribose 5-phosphate



NH



CH3



methyl

transferease



CH2



O



H



3



S



COO-



O3POCH2



CH2 CH COO+NH



CH2



homocysteine

methyltransferase



2-



from the

pentose phosphate

pathway



7



8 CH

9



N



O



H

H



9



N H CH2

CH3 NH

10



O

C



(



H COON



O



CH (CH2)2 C



)



O-



n



IMP

inosine

monophosphate

H



H



OH



OH



H



Part 7  Purines, pyrimidines and porphyrins



109



Amino acid metabolism, folate metabolism and the ‘1‐carbon pool’ II:

pyrimidine biosynthesis



55



Chart 55.1  (opposite) Biosynthesis

de novo of pyrimidines.



Amino acid metabolism and pyrimidine biosynthesis

The pyrimidine ring is derived from bicarbonate, glutamine and aspartate.

The first reaction, catalysed by carbamoyl phosphate synthetase II (CPS II),

occurs in the cytosol and produces carbamoyl phosphate from bicarbonate,

glutamine and two molecules of ATP. This is similar to the mitochondrial

reaction involved in the urea cycle, which differs in that it forms carbamoyl

phosphate from bicarbonate and NH4+ ions. Another difference is that CPS II

does not require N‐acetyl glutamate as an allosteric stimulator. The rest of the

pyrimidine ring is donated by aspartate and, after ring ­closure and oxidation,

orotate is formed. It is at this stage that phosphoribosyl pyrophosphate

(PRPP) is added to yield orotidine monophosphate (OMP), which, following decarboxylation, produces uridine monophosphate (UMP), which is the

common precursor of the pyrimidine‐­containing nucleotides (Chart 55.1).



Conversion of UMP to UTP and CTP

UMP is phosphorylated by a specific UMP kinase to form uridine

­diphosphate (UDP), which in turn is phosphorylated by the non‐specific

nucleoside diphosphate kinase to yield uridine triphosphate (UTP). When

UTP is aminated, cytidine triphosphate (CTP) is formed.



Formation of deoxycytidine triphosphate (dCTP)

and deoxythymidine triphosphate (dTTP)

dCTP is formed from cytidine diphosphate (CDP) by ribonucleotide reductase, by a mechanism analogous to the production of purine‐containing

deoxyribonucleotides (see Chapter 54).

The pathway for the formation of dTTP is quite distinct from that used to

produce deoxyadenosine triphosphate (dATP), deoxyguanosine ­triphosphate

(dGTP) and dCTP. The pathway starts with deoxycytidine diphosphate

(dCDP), which is dephosphorylated and deaminated to yield deoxyuridine

monophosphate (dUMP). This is methylated by N5,N10‐methylene tetrahydrofolate (THF), which is oxidized to dihydrofolate (DHF) in the reaction

catalysed by thymidylate synthase, and deoxythymidine monophosphate

(dTMP) is formed. The dTMP is now phosphorylated by dTMP kinase and

nucleoside diphosphate kinase to produce dTTP.

Let us return to DHF, which is formed by the thymidylate synthase reaction. This is reduced by dihydrofolate reductase, which regenerates THF.

The cycle is completed when this THF participates in the serine hydroxymethyltransferase reaction, producing glycine and N5,N10‐methylene THF;

the latter is now available once more for thymidylate synthase.



Cancer chemotherapy

Because rapidly dividing cancer cells have a great demand for DNA s­ ynthesis,

much attention has been directed at the pathways for nucleotide synthesis as

a target for chemotherapeutic intervention. These drugs are classified by

pharmacologists as ‘antimetabolites’ and fall into the following categories:

glutamine antagonists, folate antagonists, antipyrimidines and antipurines.



Glutamine antagonists



The importance of glutamine for the biosynthesis of purines and pyrimidines

has been emphasized already (see Chapter  54). Azaserine and diazo‐oxo‐­

norleucine (DON) irreversibly inhibit the enzymes involved in the glutamine‐

utilizing reactions (see Chart 54.1), reducing the DNA available to cancer cells.



Folate antagonist



N

N



H



N



CH2



O



H



COO–



N



C



N



CH



CH3



Diagram 55.1  Methotrexate.



110



1 Antipyrimidines, e.g. flurouracil. Fluorouracil inhibits thymidylate synthase and thus prevents the conversion of dUMP to dTMP.

2 Antipurines, e.g. mercaptopurine. Mercaptopurine inhibits purine biosynthesis at several stages. It inhibits PRPP amidotransferase (see Chart 54.1),

IMP dehydrogenase and adenylosuccinate synthetase (see Chart 54.2).



Salvage pathways for the recycling of purines

and pyrimidines

When nucleic acids and nucleotides are degraded, the free purine and

pyrimidine bases are formed. These can be recycled by ‘salvage pathways’

(Diagram 55.2), which require much less ATP compared with the energy‐

intensive de novo pathways shown in Charts 54.1 and 55.1. The salvage pathways for purines require specific phosphoribosyl transferases (PRTs) that

transfer PRPP in reactions analogous to that of orotate PRT (Chart 55.1).



Lesch–Nyhan syndrome

This is an extremely rare disorder caused by almost total deficiency of

hypoxanthine‐guanine PRT. In this condition, which is characterized by

severe self‐mutilation, the salvage pathway is inactive. Consequently, the

free purines hypoxanthine and guanine are instead oxidized by xanthine

oxidase to uric acid which is sparingly soluble and causes gout.



Antiviral drug azidothymidine (AZT)

AZT is an analogue of thymidine that can be phosphorylated to form the

nucleotide triphosphate, azidothymidine triphosphate (AZTTP). AZTTP

inhibits the viral DNA polymerase, which is an RNA‐dependent polymerase. The host cell’s DNA‐dependent polymerase is relatively insensitive to

inhibition by AZTTP.

Salvage pathway for purines

adenine + PRPP

adenine PRT



AMP + PPi



Lesch-Nyhan



hypoxanthine + PRPP



hypoxanthine–

guanine PRT



guanine + PRPP



IMP



+ PPi



GMP + PPi



uridine phosphorylase



uracil

ribose 1-phosphate



uridine



Pi



uridine kinase



ATP



O

(CH2)2



C



O–



thymine



thymidine phosphorylase



2'-deoxyribose

1-phosphate



Pi



thymidine



Diagram 55.2  Salvage pathways for purines and pyrimidines.



Metabolism at a Glance, Fourth Edition. J. G. Salway. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.



Metabolized

to UTP, CTP

and dTTP,

Chart 55.1



thymidine kinase



ATP



UMP



ADP



(In mammals also functions with 2'-deoxyuridine)



N



NH2



Antipyrimidines and antipurines



Salvage pathway for pyrimidines



Methotrexate, which is a close structural analogue of folate (Diagram 55.1),

inhibits DHF reductase. This prevents the reduction of DHF to THF,

as  shown in Chart  55.1. Consequently, in the absence of THF, serine

H2N



­ ydroxymethyltransferase is unable to generate the N5,N10‐methylene THF

h

needed by thymidylate synthase for dTMP production.

The clinical benefit to patients treated with high doses of methotrexate is

enhanced by the use of folinic acid, N5‐formyl THF (also known as leucovorin), which ‘rescues’ normal cells from the toxic effects of methotrexate.

Methotrexate and rheumatoid arthritis. Intermittent weekly low‐doses

of methotrexate (5–25 mg/week; cf. 5000 mg weekly for treatment of malignancy) is an important therapy for rheumatoid arthritis. The mechanism is

thought to be as shown on Chart 54.1. Methotrexate inhibits aminoimidazole‐carboxamide ribonucleoside (AICAR) transformylase causing accumulation of AICAR and its dephosphorylated metabolite, AICARiboside.

AICAR inhibits AMP deaminase and AICARiboside inhibits adenosine

deaminase causing adenosine to accumulate. Extracellular adenosine is a

potent anti‐inflammatory mediator.



ADP



dTMP



THF



(vitamin B12)



N5-methyl THF

homocysteine



CH2



NH

tryptophan



CH2



O2



S

CH3



methyl group

transferred to

acceptor, e.g.

noradrenaline



O

2

NADPH+H+



methyl

transferase



S-adenosylhomocysteine

H2O



O

2



+

NAD



HCOH

CH2OPO323-phosphoglycerate



NADH+H+



aspartate



O

-O

C



C



H2O



to

mitochondrion



C



α-ketoglutarate



H2N



3



N



2

HN 3



CH2OH



serine



N



1



COO-



oxaloacetate

glutamate



H2N



N



2

HN 3



N



H



8



1



5



4



H



7

6



9



N



CH2



COO-



CH (CH2)2 C



O



)



On



Mitochondrion



pyruvate dehydrogenase

NADH+H+



SCoA

O



CH2COOHOC



COO-



H2C



COO-



C



(



H



COO-



N



CH (CH2)2 C



O



N

8



1



4



5



O



C



ATP



H

7

6



H



C



9



O



H



C



NH



10



(



2-



O3POCH2



H



COO-



N



CH (CH2)2 C



O



H 2N



N



N



HN 3



4



5



HC COO-



8



1



2



CH2COO-



H



CH2COO-



α-ketoglutarate

dehydrogenase



CH2



NAD+ CoASH



isocitrate

dehydrogenase

Mg2+



7

6



NAD(P)H+H+

NAD(P)+



H

OH



H2O



NH4+



H2N



On



C

C



O

C



(



2-



H



COO-



N



CH (CH2)2 C



O



O3POCH2



)



On



H



N



4



5



8



1



H2N



H3+N



CH2



CH COOglutamate



histidinase



H



H

OH



4-imidazolone5-propionate



glutamate

formiminotransferase



imidazoline

propionase



2

THF



FIGLU

(N-formiminoglutamate)



HN 3



N



H

N



4



5



1



8



7

6



H

H

9



N H CH2

O

NH

HN C



O



+



N



C



ATP



N5, N10-methenyl THF



9



C



10



(



H



10



C



(



H



COO-



N



CH (CH2)2 C



O



COO-



CH (CH2)2 C



O



)



N



N



4



5



8



1



glutamine



RNA polymerase



ATP

CTP synthetase

ADP+Pi



RNA

RNA polymerase



H



7

6



N H CH2

N



O

C



10



(



H



COO-



N



CH (CH2)2 C



O



NADP+



)



On



dCTP



CDP



NADPH+H+



N5, N10-methylene THF



9



H



nucleotidase



Pi



H



H C



ribonucleotide reductase

H2O



dCDP

H2O



ATP



Pi



deoxyuridine

(dUrd)

[a plasma marker

for TS inhibition]



DNA

polymerase



ADP



DNA



dCMP

H2O



H



H 2N

On



nucleoside diphosphate kinase



UTP



On



CTP



NADP+



)



UMP

(uridine

monophosphate)



ADP



H

N



N5,N10-methylene

THF reductase



O



CH



UDP



H



7

6



H2O



O



N5-formimino-THF



5 CH

6



UMP kinase



glutamate



NADPH+H+



H2N



urocanate



HN 3



glutamine



glutamine synthetase



NH4+



2



ADP+Pi



H



OH



H



N H CH2



O

H



NH4+ ATP



N



ADP



NADPH+H+

N5,N10-methenylTHF reductase

NADP+



CH2COO-



1



O



H



CH2 CH COO+NH

3

NH

histidine



4



2



O



N10-formyl THF



9



N

2



HN 3



N5-formimino-THF

cyclodeaminase



glutamate



COO-



OMP

(orotidine

monophosphate)

orotidine monophosphate

decarboxylase

(bifunctional enzyme)



)



H



NH4+

glutamate

dehydrogenase



C



ATP



NAD+



CO2



O C COOα-ketoglutarate



H



H

OH



HN 3

H

H



H 10 N

O C

H



N5,N10-methenyl-THF

cyclohydrolase



NADH+H+



5 CH

6



O



N H CH2



O



HOCH COOisocitrate



1



N



O



H



[cis-aconitate]



H2O



2



O



THF

(tetrahydrofolate)



N H CH2



4



HN 3



H2O



H2O



PRPP



C



On



N10-formyl-THF

synthetase



ADP+Pi



H2O



H2O



COO-



orotate



CO2



aconitase



citrate



CH2



hydratase



C



O



)



H

formate



aspartate



N



5 CH

6



PPi



methotrexate

trimethoprim

aminopterin



N



O-



aconitase



NADH

H+



Q

(ubiquinone)



Hereditary orotic aciduria.

Deficiency of bifunctional enzyme



H

H2N

2

HN 3



CoA



O



NH



NADP+



COO-



CO2



QH2



FMNH2



orotate phosphoribosyl

transferase (PRT)

(bifunctional enzyme)



DHF

(dihydrofolate)



10



dihydrofolate

reductase



CH2



O C SCoA

succinyl CoA



(



H

N



1



N

H



methotrexate

trimethoprim

aminopterin



NADPH+H+



Krebs cycle



CH2COO-



dihydroorotate



H



Cytosol



aspartate

aminotransferase

α-ketoglutarate



COO-



C



NADP+



glycine



acetyl CoA



H3+NCH



O



NH



dihydrofolate

reductase



H3+NCH2



NAD+



citrate

synthase



4



2



O



CH2



NADPH+H+



O



H2O



9



N



C



folate



10



N 5,N 10-methylene THF



pyruvate

carrier



C O

H2C COO-



7

6



5



4



O



THF



H



8



O



COO-



CH

COO-



C



H +NCH



serine

hydroxymethyl

transferase



5 CH2

6



FMN



HN 3



pyruvate



C



CH

COO-



carbamoyl aspartate



dehydrogenase

(mitochondrial)



O



CH3



H3C



1



N

H



phosphatase



C O



HCO3-



4



2



O



COO-



pyruvate

kinase

Mg2+ K+



CO2



5 CH2

6



O



HN 3



Pi



COO-



ADP+Pi



1



N

H



carbamoyl aspartate

dihydroorotase



COO-



glutamate



H O

2



CH2

phosphoenolpyruvate



thiamine PP

lipoate

riboflavin



4



2



O



3-phospho

serine



COPO32-



carboxylase

(biotin)



C



H2N 3



C



3-phosphoserine

r

α-ketogluta

-ketoglutarate

k

r

rate

aminotr

transfe

f rase

r

aminotransferase



COO-



ATP



aspartate transcarbamoylase

(ACTase)



reductase



NAD+



3-phospho

hydroxypyruvate



dehydrogenase



enolase

Mg2+



CoASH



glutamate 2ADP+Pi



glutamine



NADH+H+



CH2OH

2-phosphoglycerate



A

ATP



2ATP



2



α-ketoadipate



HCOPO32-



ADP



O

O

OH 5-phosphoribosyl

pyrophosphate (PRPP)



O



COO-



H O

2



OH



H O

2

dehydrogenase



-OOC(CH )

2 3



phosphoglycerate

r

Mg2+ mutase



O-



P O P O



C

O

OPO32carbamoyl phosphate



NH +

4



NAD+



O-



O



H2N 3



2-aminomuconate



to

mitochondrion



COO-



bicarbonate



2-aminomuconate

semialdehyde



phosphoglycerate

r

kinase



A

ATP



HCO



carbamoyl phosphate

synthetase II (CPS II )



CO2



deaminase



CH2OPO321,3-bisphosphoglycerate

ADP





3



picolinate

decarboxylase

o



+

NADH+H



glycolysis



H

H



feedback

inhibition by UTP



3,4-dioxygenase

3,4-dio

ioxygenase



2-amino-3-carboxymuconate

semialdehyde



α-ketobutyrate



HCOH



formate



O

H



kyn

y ureninase

kynureninase



homoserine



O C OPO32-



H



3-hydroxyanthranilate



H2O

llyase

ly

yase



+

NH

4



H



3-monooxygenase

o



H O

2



cystathionine

cysteine



C



O3POCH2



alanine



cystathionine

synthase



H O

2



ribose 5-phosphate



3-hydroxykynurenine



adenosyl

homocysteinase



homocysteine



methionine

salvage

pathway



OH



+

NADP



adenosine



serine



OH



AMP

2-



OO



kynurenine



OH



H



ribose phosphate

pyrophosphokinase



formamidase



adenosyl

transferase



Pi+PPi



S-adenosylmethionine



H



H



ATP



2,3-dioxygenase



H2O



H2O



O



H



N-formylkynurenine



methionine

ATP



O3POCH2



from the

pentose phosphate

pathway



3



3



homocysteine

methyltransferase



2-



CH2 CH COO+NH



COOH +NCH



N

2



HN 3



1



4



O



8



5



NH4+



H



N

7

6



H

9



N H CH2

CH3 NH

10



C



(



H



COO-



N



CH (CH2)2 C



O



)



On



phosphatase



Pi



thymidylate synthase (TS)



dUMP



N5-methyl THF

O



deoxycytidine

deaminase



DNA

polymerase



N5, N10-methylene THF



dTMP



fluorouracil



dTTP



dTDP

ATP



ADP



ATP



ADP



DHF



Part 7  Purines, pyrimidines and porphyrins



111



Krebs uric acid cycle for the disposal of nitrogenous waste



56



Krebs and his trinity of cycles

The distinguished biochemist from Oxford University, Sir Hans Krebs

(1900–1981), discovered three biochemical cycles which have been

described elsewhere in this book. Earlier we met the Krebs citric acid cycle

or TCA cycle, referred to in this book simply as Krebs cycle (see Chapter 19),

and a truncated version of this, the glyoxylate cycle (see Chapter 20). Also,

we have seen how mammals dispose of their toxic nitrogenous waste from

amino acid catabolism by using the ornithine or urea cycle (see Chapter 51)

to form urea for urinary excretion. Hans Leo Kornberg wrote in 2000,

‘Everyone who has taken biology at school has heard of the Krebs cycle, but

few realize that Krebs also discovered two other cycles.’ It is remarkable that

even Kornberg, ironically co‐discoverer of the glyoxylate cycle, had overlooked a fourth Krebs cycle which was published in 1978.



A fourth Krebs cycle in uricotelic animals

There is a fourth Krebs cycle which has been almost totally overlooked by

text books and biochemists. This is the uric acid cycle, which operates in

birds and probably in other uricotelic animals (e.g. land reptiles) and

insects. The term uricotelism is used to describe animals that dispose of

their ­nitrogenous waste from protein metabolism as uric acid. Similarly, the

terms ureotelism and ammonotelism apply to the excretion of nitrogen as

urea and ammonia respectively. NB: It should be emphasized that whereas

mammals dispose of nitrogenous waste from protein catabolism as urea

(see Chapter  51, they dispose of purine waste as uric acid or allantoin

(Diagram 56.1).

Diagram 56.1  Metabolism of uric

acid to ammonium ions. This

sequence of enzymatic reactions is

mainly (but not entirely) contained

within the intestinal flora of the

species. For example, uricase is

present in the liver of mammals with

the notable exception of the primates.

Therefore, this diagram should be

used with judicious caution as the

excretion of nitrogenous waste is

complicated by many anomalies in

response to environmental

adaptation. It is a fascinating topic for

the study of evolutionary

biochemistry.



Origin of the nitrogen used for uric acid synthesis

Nitrogen from the catabolism of amino acids is incorporated into glutamate

or converted to ammonium ions by a process analogous to that used for the

urea cycle (Chart 56.1). Ammonium ions are very toxic and react with glutamate to form glutamine. Uric acid contains four nitrogen atoms: two of

these are derived from glutamine, one from glutamate and one from glycine.

The uric acid cycle is a cyclic adaptation of the process used for mammalian

purine catabolism shown in Chapter 54. Inosine monophosphate (IMP) is

formed, which reacts with pyrophosphate to form hypoxanthine and 5‐

phosphoribosyl pyrophosphate (PRPP). This reaction is catalysed by

hypoxanthine phosphoribosyl transferase (EC 2.4.2.8), otherwise known

as IMP pyrophosphorylase. The phosphoribosyl transferases (PRTs) have



been referred to in Chapter 55 in connection with the salvage pathway for

recycling purines and pyrimidines. Pyrophosphate is regenerated by the

PRPP amidotransferase reaction. NB: The substrate that is recycled is

PRPP, so strictly speaking this should be called the ‘PRPP cycle for the production of uric acid’.



Energy considerations

Formation of uric acid by the cycle requires hydrolysis of 6‐phosphoanhydride bonds (see Chapter  2. The reactions are: glutamine synthetase (2

bonds); FGAM synthetase (1 bond); AIR synthetase (1 bond); SAICAR synthetase (1 bond); and GAR synthetase (1 bond). Thus for the excretion of

four nitrogen atoms as uric acid, six phosphoanhydride bonds are hydrolysed, which is equivalent to 1.5 per nitrogen atom. This compares favourably with excretion of urea by mammals where two phosphoanhydride bonds

are used for each nitrogen atom synthesized.

What about glycine?  When considering the energy for the biosynthesis

of uric acid it is easy to overlook that a molecule of glycine is incorporated. If

this glycine was oxidized as fuel it would generate ATP as follows. Chart 46.2

shows that glycine can be metabolized to pyruvate and oxidized in Krebs

citric acid cycle. The following NADH + H+‐dependent reactions – pyruvate

dehydrogenase, isocitrate dehydrogenase, α‐ketoglutarate dehydrogenase

and malate dehydrogenase  –  generate a total of 4 NADH + H+ which,

assuming a P/O ratio of 2.5 (see Chapter  6), yield 10 ATP. Succinyl CoA

synthetase produces 1 GTP (equivalent to ATP) and succinate dehydrogenase

produces 1 FADH2 yielding 1.5 ATP. Thus a glycine molecule has the

potential to produce 12.5 molecules of ATP.)



Ammonotelic, uricotelic and ureotelic animals

Although all animals have the challenge of disposing toxic ammonia from

protein catabolism, there are three main nitrogenous waste products:

ammonium ions, uric acid and urea. For animals such as bony fish and

larval amphibians, which inhabit an aquatic environment with an

­unlimited supply of water, ammonia is the principal excretory product.

Approximately 400 ml of water is needed to excrete 1 g of ammonia.

However, terrestrial animals such as insects, terrestrial reptiles and birds,

can tolerate a dry environment and excrete uric acid requiring only

approximately 8 ml of water per gram of nitrogen – whereas urea needs

40 ml of water.



NH2

O



H

N



C

C



HN

C

O



C



N

H



H

N



O

C



O



N

H



uric acid

(keto-tautomer)



2H2O



H2O2 CO2



H2N

C



O2



Primates

Uric acid is the excretory

product of purine catabolism

(Chapter 54).

Nitrogenous waste from amino

acids (proteins) is excreted as

urea made by the Ornithine

(Urea) Cycle (Chapter 51)



uricase



O



6



1

2

3



N

H



COOH



5C

4C



7

8C



O



2H2O



C



9



N

H



O

H2N



allantoinase



allantoin



Mammals other than primates

Allantoin is the excretory

product of purine catabolism

(Chapter 54).

Nitrogenous waste from amino

acids (proteins) is excreted as

urea made by the Ornithine (Urea)

Cycle (Chapter 51)



O



N

H



OH

C H NH2

C

C

N

H



H2O



NH2



O



CHO



NH2



2H2O



2CO2



O



allantoic acid



O2



O



allantoicase



NH2



2 urea



Elasmobranchs

(cartilaginous fish) and amphibia

Urea made by the Ornithine (Urea)

Cycle (Chapter 51) is the

excretory product of

nitrogenous waste from

amino acids (proteins)



Metabolism at a Glance, Fourth Edition. J. G. Salway. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.



+



NH4



C



Birds, reptiles, insects

Uric acid (made by the uric acid

cycle) is the excretory product of

purine metabolism AND of

nitrogenous waste from amino

acids (proteins)



112



C



urease



+



NH4

+



NH4



+



NH4



4 ammonium ions



Teleosts (bony fish),

marine invertebrates

and microorganisms

Ammonia is the excretory

product of purine catabolism

AND of nitrogenous waste

from amino acids (proteins).

Especially important for

microorganisms in the

digestive tract of ruminants



Transamination reactions



Deamination reactions

alanine



arginine



tryptophan



lysine

α-KG



glycine



phenylalanine



isoleucine



valine



alanine



tyrosine



aspartate



cysteine



α-ketoglutarate



α-ketoglutarate



alanine

aminotransferase



aminotransferase



alanine



α-KG



leucine aspartate



serine



histidine

glutamate

ornithine α-aminoadipate

proline



mitochondrion



oxaloacetate



pyruvate



pyruvate



α-ketoadipate



3-hydroxypyruvate



acetyl CoA

4-hydroxyphenylpyruvate



NADH+H+



α-ketoglutarate



NAD+



α-ketoacid



NH4+



aspartate



α-ketoacid

glutamate



glucose



NH4+



NH4+



NH4+



methyl

glyoxal



pyruvate



asparagine



methionine



NH4+



NH4+



alanine



glutamate



α-ketoacid

(oxaloacetate)



threonine

serine



pyruvate



aminotransferase



glutamate



branched chain

α-ketoacids



saccharopine

guanidino

-acetate



creatine



branched-chain amino acids



pyruvate



aminotransferase

glutamate



succinyl CoA



glutamate



glutamate



α-ketoglutarate



succinyl CoA



α-ketoacid

(oxaloacetate)



glycogen



glutamate

dehydrogenase

COO-



NH4+

ammonium

ion



COO-



ATP



H3+N CH



glutamine

synthetase



CH2 COO-



oxaloacetate



CH2



α-ketoglutarate



glutamate aspartate

aminotransferase



glutamate



H3+N CH



H3+N CH



CH2



COO-



CH2 COO-



COO-



O CH



CH2



CH2



ADP+Pi



COO-



COO-



O CH



COO-



aspartate



CH2

CH2 CONH2



glutamine

3



H 2C 5

C



HN

glutamate



2-



ATP



H2C 5



O



NH



H



H



OH



OH



2-



H



N

O



O3POCH2

H



formylglycinamidine

ribonucleotide (FGAM)



AIR

carboxylase



9



H2N



AIR

synthetase



O



H2O



8 CH



N



C

6



-O



8 CH



3 4C



8 CH



9



7



5C



5C



H

OH



8 CH



3 4C



HC



ATP



O



ADP+P



H

H



aminoimidazole

ribonucleotide (AIR)



COO-



9



N



O3POCH2



H

OH



SAICAR

synthetase



7



H2N

2-



H



H



H



OH



OH



N



C



1



NH



CH2



i



succinylaminoimidazole

carboxamide

ribonucleotide

(SAICAR)



O

6



7



5C



8 CH



3 4C



adenylosuccinase

(ASase)



9



H 2N

COO-



N



2-



O



O3POCH2



carboxyaminoimidazole

ribonucleotide (CAIR)



ADP+P

i



H

N



7



H



7



4



O



O3POCH2



ADP+P

i



ATP



CO2



N



H



H

N



H



H



H



H



OH



OH



O



FGAM synthetase



fumarate



2-



3



OH



GAR

transformylase

THF



N10-formyl

THF



O-



7



O



C4



2-



H



H



5



9



NH



O



O3POCH2



CH2 NH2



H



HO



GAR

synthetase



2-



O-



OHO



O



O-



O



P O P OH

O



pyrophosphate



AICAR

transformylase

O



O



pyrophosphate



HN 1



ATP

2-



O



O3POCH2



H



glycinamide OH OH

ADP+Pi

ribonucleotide (GAR)



H



HC 2



NH2



H



glutamate

H2O



H



H



N5,N10-methylene

THF

COOH +N CH

3



2



glycine



O3POCH2

H



H



O

H



H



OH



OH



O



O-



2–



O-



P O P O



O



hypoxanthinephosphoribosyl

transferase



O



5-phosphoribosyl

pyrophosphate

(PRPP)



COO-



COO-



CH2OH



3



5C

4C



6



H2N



7



O CH



8 CH



2



9



O3POCH2

H



7



3



8 CH



4C



N

O



O3POCH2

H



H



OH



OH



H



IMP

cyclohydrolase

H



H



THF



H



H



OH



OH



H



N10-formyl

THF



9



NH

2-



N



O

H



N

5C



H



PRPP

amidotransferase



H3+N CH



THF



6



N

2-



OH OH

β 5-phosphoribosylamine



N



C



C



1



H



8 CH



O



O3POCH2



P O P OH

O



7



5C



aminoimidazole-carboxamide

3 4C

9

ribonucleotide (AICAR)

H2 N

N



Uric acid cycle



2



OH



6



H 2N



C4 9 O

NH

O

O POCH

formylglycinamide

H H

H H

ribonucleotide (FGAR)

O



N



C



1



formaminoimidazole-carboxamide

ribonucleotide (FAICAR)



H2O



OH OH

inosine

monophosphate



H3+N CH



serine



serine

hydroxymethyl

transferase



CH2

CH2 CONH2

O



glutamine



N



C

HN 1

HC 2



6



3



5C

4C



7



8 CH

9



N

H



N



hypoxanthine

H2O



O2

xanthine

oxidase



Birds, reptiles, insects

Uric acid (made by the uric

acid cycle) is the excretory

product of purine metabolism

AND of nitrogenous waste

from amino acids (proteins)



H2O2



O



H

N



C

HN



C



C



C



O



N

H



O



O



N

H



uric acid

(keto-tautomer)



Although other biochemists established the intermediates involved in purine

metabolism as a linear process, it was Mapes and Krebs who organized the

pathway as a cycle. The Krebs uric acid cycle described here is in their ­publication

cryptically entitled: ‘Rate‐limiting factors in urate synthesis and gluconeogenesis

in avian liver.’



N



C



C



HN 1

C



H2O2



H2O

xanthine

oxidase



O



6



2

3



N

H



5C

4C



7



8 CH

9



N

H



xanthine



Chart 56.1  Krebs uric acid cycle for

the disposal of nitrogenous waste.



O2



References



Kornberg H. (2000) Krebs and his trinity of cycles. Nature Rev Cell Biol, 1,

225–8.

Mapes J.P., Krebs H.A. (1978) Rate‐limiting factors in urate synthesis and

gluconeogenesis in avian liver. Biochem J, 172, 193–203.



Part 7  Purines, pyrimidines and porphyrins



113



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Chart 51.1: nitrogen, in the form of ammonium ions or glutamate, is used for urea synthesis

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