1. Year Two Review
Eric Niederhoffer, Ph.D.
SIU-SOM

2. Outline Pyrimidine and purine synthesis Glycogen storage disorders
including salvage and degradation
Glycogen storage disorders
Lysosomal storage disorders
Heme synthesis and degradation
including oxygen binding/unloading of heme
Integration of metabolism
including lipid synthesis/degradation, glycolysis/gluconeogenesis, TCA cycle and glycogenolysis/glycogen synthesis

3. Pyrimidine and Purine Synthesis
HCO3- + Gln CP
CPSII
R5P
PRPP
RPK
Asp
Oro
UMP
UMPS
IMP
Gln
Gly
CO2
Asp
N10fTHF
UTP
RNA
CDP
dCDP
dUMP
DNA
GDP
ADP
Gln: glutamine CP: carbamoyl phosphate Oro: orotic acid UMP: uridine monophosphate
UTP: uridine triphsophate CDP: cytosine diphosphate dCDP: deoxycytosine diphosphate
dUMP: deoxyuridine diphosphate dTMP: deoxythymidine monophosphate
DNA: deoxyribonucleic acid RNA: ribonucleic acid R5P: ribose-5-phosphate
PRPP: 5-phosphoribosylpyrophosphate Gly: glycine N10fTHF: N10-formyltetrahydrofolate
Asp: aspartate IMP: inosine monophosphate GDP: guanosine diphosphate
ADP: adenosine diphosphated dGTP: deoxyguanosine triphosphate
dATP: adenosine triphosphate
CPSII: cytosolic carbamyolphosphate synthetase, feedback inhibition by UTP
UMPS: UMP synthase, a bifunctional enzyme (orotate phosphoribosyltransferase, OMP decarboxylase), deficiency of either activity leads to hereditary orotic aciduria
TS: thymidylate synthase, inhibited by fluorouracil
RR: ribonucleotide reductase, inhibited by hydroxyurea
Folic acid and its derivatives (for example, THF) are important for biosynthesis and salvage pathways. TS
N5,N10-mTHF
dTMP
dGTP
dATP RR

4. Pyrimidine and Purine Salvage
UTP
CDP
dCDP
dUMP TS
N5,N10-mTHF
dTMP
RNA
DNA
IMP
GDP
ADP
dGTP
dATP RR U T
PRPP
UMP
TMP RR
UTPT A
APT G
PRPP
HGPT
adenosine
inosine
ADA
UTP: uridine triphosphate CDP: cytosine diphosphate dCDP: deoxycytosine diphosphate
dUMP: deoxyuridine monophosphate dTMP: deoxythymidine monophosphate
IMP: inosine monophosphate GDP: guanosine diphosphate ADP: adenosine diphosphate
dGTP: deoxyguanosine triphosphate dATP: deoxyadenosine triphosphate
UMP: uridine monophosphate TMP: thymidine monophosphate RNA: ribonucleic acid
DNA: deoxyribonucleic acid U: uridine T: thymine A: adenine G: guanine
N5,N10-mTHF: N5,N10-methylenetetrahydrofolate ADA: adenosine deaminase
PNP: purine nucleotide phosphorylase HGPT: hypoxanthine-guanine phosphoribosyl transferase
APT: adenine phosphoribosyl transferase X: xanthine
XO: xanthine oxidase, inhibited by allopurinol
There is feedback inhibition of nucleotide synthesis by products
Folic acid and its derivatives are important for biosynthesis and salvage pathways. HX
PNP X XO
urate XO

5. Pathway Disorders Rare autosomal recessive disorders
UMP synthase – deficiency in either orotate phosphoribosyltransferase or OMP decarboxylase leads to hereditary orotic aciduria, megaloblastic anemia appearing weeks to months after birth that does not respond to cobalamin, folic acid, or iron, orotic crystalluria and nephropathy, cardiac malformations, strabismus, and recurrent infection. Urine orotic acid overexcretion. Enzyme assay of RBC. Treatment with oral uridine.
Adenosine deaminase – (Severe combined immunodeficiency disorder) variety of clinical phenotypes, history of infections, diarrhea, dermatitis, and failure to thrive, ribs and vertebrae abnormalities (defects in cartilaginous structures). Lymphopenia, B and T cell production affected. Enzyme assay of RBC/WBC. Treatment by bone marrow/stem cell transplantation or enzyme replacement.
Purine nucleotide phosphorylase – (Immunodeficiency) lymphopenia, thymic deficiency, recurrent infections, and hypouricemia, developmental delay, ataxia, or spasticity. T cell production affected. Enzyme assay of RBC, lymphocytes, fibroblasts. Treatment by bone marrow/stem cell transplantation.
Adenine phosphoribosyl transferase – frequent infections, renal colic, renal failure. Elevated urine levels of 2,8-dihydroxyadenine, 8-hyroxyadenine, and adenine; serum uric acid normal. Enzyme assay. Treated with dietary purine restriction, high fluid intake, and avoidance of urine alkalinization, Allopurinol to prevent oxidation of adenine.
Screening newborn for Severe combined Immunodeficiency disorder (SCID) is by PCR and T cell receptor rearrangement excision circles (TREC) decrease, surrogate marker for thrombopoiesis.
ADA deficiency: ADP, GTP, ATP and 2’deoxyadenosine increase in lymphocytes, 2-dAd inhibits S-adenosyl-L-homocysteine (SAH) hydrolase, SAH increase inhibits all methylation reactions. dATP increase to toxic levels can inhibit ribonucleotide reductase. Ad increase leads to 2,8-dihydroxyAd and b-hydroxyAd in urine.
PNP deficiency: dATP and dGTP increase, dGTP inhibits ribonucleotide reductase

6. Pathway Disorders X-linked recessive disorder
Hypoxanthine-guanine phosphoribosyl transferase – (Lesch-Nyhan syndrome) usually presents at 3 to 12 months with orange sandy urine precipitate, dystonia, intellectual disability, self-mutilation (lips, tongue, fingers), and gout. Elevated serum and urine uric acid levels. Enzyme assay on RBC, lymphocytes, fibroblasts. Molecular genetics of gene. Treated supportively with low-purine diet, allopurinol, and plenty of hydration.

7. Glycogen Storage DisordersGS
GSD0
Glycogen
G1P
G6P
Glc
UDP-Glc
F6P
F16BP
acid maltase
GSDII ls
transglycosylase
branching enzyme
GSDIV
hPP
GSDVI
mPP
GSDV
debranching enzyme
GSDIII
hG6Pase
GSDI
GSD: glycogen storage disease hPP: hepatic phosphorylase mPP: muscle phosphorylase
PPK: phosphorylase kinase hG6Pase: hepatic glucose-6-phosphatase
UDP-Glc: uridine diphosphate glucose G1P: glucose-1-phosphate Glc: glucose
F6P: fructose-6-phosphate F16BP: fructose-1,6-bisphosphate
PFK-1: phosphofructokinase-1 debranching enzyme: a(1→6)Glcase
branching enzyme: amylo-(1,4 to 1,6) transglycosylase
GSD type 0 GSD type I:von Gierke GSD type II: Pompe GSD type III: Cori
GSD type IV: Andersen GSD type V: McArdle GSD type VI: Hers
GSD type VII: Tarui GSD type IX: GSD type XI: Fanconi-Bickel hepatorenal glycogenosis
II, III, V, and VII cause muscle weakness
PFK-1
GSDVII

8. Pathway Disorders Rare autosomal recessive disorders
Glycogen synthase –.(GSD type 0) fasting hypoglycemia, ketosis, especially before feeding. Periodic acid-Schiff stain shows decreased hepatic glycogen stores, muscle is normal. Treatment is appropriate diet to avoid hypoglycemia.
Glucose-6-phosphatase – (GSD type Ia, Von Gierke) history hypoglycemic seizures, hypotonia, hepatomegaly, xanthomas, manifestations of gout, hypertension, renal failure, and short stature. Fasting glucose, ischemic forearm test (negative), Enzyme assay. Treatment by high-protein diet, uncooked corn starch.
Lysosomal acid maltase – (GSD type II, Pompe, a-1,4-glucosidase); infantile - feeding and breathing difficulties, hypotonia, cardiomegaly; adult – limb-girdle weakness, respiratory muscle involvement. Hyperlipidemia, fasting ketonemia. Ischemic forearm test normal. Enzyme assay of fibroblasts. Periodic acid-Schiff stain is positive for lysosomal glycogen inclusions. Treatment by enzyme relacement, high protein diet.
Debranching enzyme – (GSD type III, Forbes-Cori, amylo-1,6-glucosidase), infantile seizures, hepatomegaly, growth retardation, progressive muscle weakness. Ischemic forearm test positive. Enzyme assay of fibroblasts. Periodic acid-Schiff stain is positive for basophilic glycogen deposits in all tissues. Treatment is supportive by corn starch, liver transplantation.

9. Pathway Disorders
Rare autosomal recessive disorders
Branching enzyme – (GSD type IV, Andersen, transglucosidase) history not specific, hepatic failure, cirrhosis, hepatosplenomegaly, failure to thrive. Prenatal PCR and DNA analysis. Enzyme assay. Diffuse amylopectin-like deposits in the heart, liver, muscle, spinal cord, and peripheral nerves. Treatment is supportive with liver transplantation and diet.
Myophosphorylase – (GSD type V, McArdle) cramps, fatigue, and pain after exercise (depends on severity of deficiency), unique "second-wind" phenomenon. Ischemic forearm test positive. Enzyme assay. Periodic acid-Schiff stain gives subsarcolemmal blebs. Treatment by avoiding intense exercise, provide high protein diet.
Hepatic phosphorylase – (GSD type VI, Hers) most common among Mennonite religious group, also X-linked form, history of bulging abdomen, growth retardation, and slight delay in motor milestones, hepatomegaly. Enzyme assay on liver biopsy, RBC, WBC; molecular genetics of gene. Glycogen-distended hepatocytes, muscle normal. Treatment with dietary management as appropriate for clinical presentation.
Phosphofructokinase-1 – (GSD type VII, Tarui, M form, classic) history of exertional fatigue, nausea and vomiting, muscle cramps, hyperuricemia, myoglobinuria following high-intensity exercise. Ischemic forearm test positive. Enzyme assay on muscle biopsy. Periodic acid-Schiff diastase-negative stain gives subsarcolemmal blebs. Treatment to avoid high carbohydrate diet especially before exercise.

10. Lysosomal Storage Disorders
Lipid metabolism
Landing, Sandhoff, Tay-Sachs, Krabbe, Gaucher, Niemann Pick (A,B), Wolman, metachromatic leukodystrophy, Fabry
Glycoprotein metabolism
Schindler
Mucopolysaccharide metabolism
Hurler/Scheie, Hunter, Sanfilippo (A,B,C,D), Morquio (A,B), Maroteaux–Lamy, Sly
Other lysosomal enzymes
Pompe, Niemann-Pick (C)
The following has a nice review on lysosomal storage diseases:

11. Oligosaccharidoses a-Mannosidosis
a-Mannosidase
GM2 gangliosidosis variant O (Sandhoff-Jatzkewitz disease)
b-N-Acetylhexosaminidases A&B
Aspartylglycosylaminuria
4-L-Aspartylglycosylamine amidohydrolase 6 5
NANA
Gal
GlcNAc 4 a b b
Man 6 5
NANA
Gal
GlcNAc 4 3 a b a 7 2 1 6 5 a
Man
GlcNAc
GlcNAc
Asn
NANA
Gal
GlcNAc 4 3 b b a b 8 b
Man
Fucosidosis
a-Fucosidase 6 5
GlcNAc
Fuc
b-Mannosidosis
b-Mannosidase
NANA
Gal
GlcNAc 4 a b
Mucolipidosis I (Sialidosis)
Sialidase
GM1 gangliosidosis
b-Galactosidase
Typical Asn-GlcNAc OS structure
NANA: N-acetylneuraminic acid (sialic acid) Gal: galactose
GlcNAc: N-acetylglucosamine Man: mannose Fuc: fucose
Asn: asparagine OS: oligosaccharide

12. Glycosaminoglycoses (mucopolysaccharidoses)
Mucolipodosis VII
b-glucuronidase
Hunter’s
iduronate sulfatase
Sanfilippo’s A
heparan N-sulfatase
Hurler-Scheie
a-L-iduronidase
Aldurazyme®
(laronidase)
Sanfilippo’s C
Acetyl-CoA: a-glucosaminide acetyltransferase
Maroteaux-Lamy
N-acetylgalactosamine sulfatase 2 4 5
Sandhoff/Tay-Sachs
b-hexosaminidase A,B,S DS
IdUA
GalNAc
GlcUA
GalNAc a b b b 1 3
OSO3H
OSO3H
OSO3H 2
7,9
Sanfilippo’s B
N-acetylglucosaminidase 5 9 HS
IdUA
GlcN
GlcUA
GlcNAc a a b a 1 6 8
OSO3H
OSO3H
Sanfilippo’s D
N-acetylglucosamine-6-sulfatase
OSO3H
IduUA: iduronic acid GalNAc: N-acetylgalactosamine
GlcUA: glucuronic acid GlcNAc: N-acetylglucosamine GlcN: glucosamine
Gal: galactose DS: dermatan sulfate HS: heparan sulfate
KS: keratan sulfate 11 4 KS
Gal
Morquio’s B
b-galactosidase
GlcNAc
Gal
GlcNAc b b b b 10 8
Morquio’s A
N-acetylgalactose-6-sulfatase
OSO3H
OSO3H
OSO3H

13. Gangliosidoses GD1 GM1 GM2 GM3 neuraminidase (sialidase) Cer Gal Glu
NANA
GalNAc b
Generalized gangliosidosis
b-galactosidase
GM1
Cer
Gal
Glu
NANA
GalNAc b
GM2
Cer
Gal
Glu
NANA
GalNAc b
Tay-Sachs disease
b-hexosaminidase A
GM2 activator
GM3
Cer
Gal
Glu
NANA b
Sialidosis
neuraminidase
(sialidase)
SAP-B
Cer
Gal
Glu b
Fabry’s disease
a-galactosidase A
SAP-B
b-galactosidase
SAP-B, SAP-C
Cer
Gal
Glu a b
Cer
Glu b
Sandhoff’s disease
b-hexosaminidase A&B
Gaucher’s disease
b-glucosylceramidase
SAP-C
Cer
Cer
Gal
Glu
GalNAc a b
NANA: N-acetylneuraminic acid (sialic acid) Cer: ceramide
Glu: glucose Gal: galactose GalNAc: N-acetylgalactosamine
G: ganglioside D1: dineuraminic acid + Gal-GalNAc-Gal-Glc-Cer
M1: neuraminic acid + Gal-GalNAc-Gal-Glc-Cer
M2: neuraminic acid + GalNAc-Gal-Glc-Cer M3: neuraminic acid + Gal-Glc-Cer
SAP: sphingolipid activator protein Cer-Glu-Gal: lactosylceramide
Cer-Glu: glucosylceramide S: sphingosine FA: fatty acid
Cer-Glu-Gal-Gal-GalNAc: globoside Cer-Glu-Gal-Gal: globotriaosylceramide
PC: phosphocholine Cer-PC: sphingomyelin
Cer-Gal-SO3H: sulfatide Cer-Gal: galactosylceramide
Krabbe’s disease
b-galactosylceramidase
SAP-A, SAP-C
Cerezyme
Cer
Gal b
Cer PC
Niemann-Pick disease
sphingomyelinase
S + FA
Metachromatic leukodystrophy
arylsulfatase A
SAP-B
Cer
Gal
SO3H b

14. General Physical Features
Coarse facial features (sometimes with macroglossia)
Corneal clouding or related ocular abnormalities
Angiokeratoma
Umbilical/inguinal hernias
Short stature
Developmental delays
Joint or skeletal deformities
Organomegaly (especially liver and spleen)
Muscle weakness or lack of control (ataxia, seizures, etc.)
Neurologic failure/decline or loss of gained development
The following has useful information:

15. Heme Synthesis mit SCoA + Gly 5AS 5ALA PBG PBGS PBGD HMB FC Heme
UPGIIIS
UPGIII
PPIX
PPGO
UPGIIIDC
CPGIII
MIT: mitochodrion SCoA: succinyl CoA Gly: glycine
5ALA: 5-aminolevulinic acid PBG: porphobilinogen
5AS: 5-aminolevulinic acid synthase; feedback inhibition by heme and glucose, regulates transcription, mRNA stability, 5AS import; positive regulation by EtOH, sex steroids, barbituates, sulphonamides, anticonvulsants
PBGS: PBG synthase (ALA dehydratase), very rare deficiency AR, inhibited by styrene, lead, trichloroethylene, and bromobenzene; hereditary tyrosinemia leads to accumulation of succinylacetone, an inhibitor.
PBGD: PBG deaminase: deficiency AD (acute intermittent porphyria)
HMB: hydroxymethylbilane UPG: uroporphyrinogen
UPGIIIS: UPG III synthase, deficiency AR (congenital erythropoetic porphyria, Günther disease)
UPGIIIDC: UPGIII decarboxylase, deficiency mostly acquired (AD), alcohol abuse (porphyria cutanae tarda)
CPG: coproporphyrinogen CPGO: CPG oxidase, deficiency AD (hereditary coproporphyria)
PPGIX: protoporphyrinogen IX PPGO: PPG oxidase, deficiency AD (variegate porphyria)
FC: ferrochelatase, deficiency AD/AR/X-linked (erythropoetic protoporphyria), lead poisoning
CPGO
PPGIX

16. Pathway Disorders
PBG synthase – (5-aminolevulinic acid dehydratase) extremely rare autosomal recessive (hepatic porphyria) neurological findings, abdominal tenderness, neuropathy, not associated with cutaneous photosensitivity. Elevated urine ALA, coproporphyrin III and protoporphyrin IX, normal PBG, elevated RBC zinc protoporphyrin but decreased (80%) PBG synthase. DNA analysis. Treatment by avoiding precipitating factors, drugs that induce P450 induction, provide hematin, high carbohydrate diet (glucose inhibits 5-AS).
PBG deaminase – (Acute intermittent porphyria) autosomal dominant, abdomen pain, psychiatric symptoms (hysteria), motor neuropathies (more commonly lower limbs), and constipation but no skin rash. Increased urinary porphobilinogen secretion, molecular genetic analysis. Treatment during attacks with high carbohydrate (glucose) diet and hematin, otherwise, balanced diet.
Uroporphyrinogen III synthase – (Congenital erythropoetic porphyria, Gunther disease) rare autosomal recessive, photosensitivity, nail abnormalities, brown or pink teeth. Elevated urine and RBC levels of uroporphyrin I, hemolytic anemia. Enzyme assay, molecular genetic analysis, red porphyrin fluorescence in intact RBC and erythroid precursor cells. Treated with absolute avoidance of sun exposure, supportive/cosmetic care.
Uroporphyrinogen III decarboxylase – (Porphia cutana tarda) 80% acquired/20% familial/autosomal dominant, acquired by ethanol abuse, estrogen therapies, hemochromatosis genes, hepatitis and human immunodeficiency viral infections, environmental toxins, photosensitivity, tea/wine colored urine. Carboxylated porphyrins in serum and urine. Enzyme assay of RBC, molecular genetic analysis. Treatment with avoidance of sunlight/environmental exposure.

17. Pathway Disorders
Coproporphyrinogen oxidase – (Hereditary coproporphyria) autosomal dominant, abdominal pain, neuropathies (motor, lower limbs), constipation, and skin changes (photosensitivity). Excess secretion and levels of coproporphyrins in stool and urine. Treatment with high carbohydrate (glucose) diet and hematin.
Protoporphyrinogen oxidase – (Variegate porphyria) autosomal dominant, photosensitivity, abdominal discomfort. Urinary aminolevulinic acid and porphobilinogen levels are greatly elevated during attacks, molecular genetic analysis. Treatment with avoidance of inducing drugs, providing high carbohydrate diet, hematin.
Ferrochelatase – (Erythropoetic protoporphyria) autosomal dominant (X-linked, autosomal recessive), photosensitivity, heptabiliary disease, jaundice. Elevated protoporphyrin concentration in red blood cells, plasma, bile, and feces. Treatment with avoidance of sun exposure, maintain balanced diet.

18. Heme Degradation res indirect unconjugated pre-hepatic HO BV Heme BVRBR
albumin
albumin-BR
ligandin
albumin
ligandin-BR
hepatocyte
BR diglucuronide
UDP-GT ER
direct
conjugated
post-hepatic
RES: recticuloendothelial system HO: heme oxidase BV: biliverdin
BVR: biliverdin reductase BR: bilirubin
UDP-GT: uridinediphosphate-glucuronyl transferase, deficiency mild (Gilbert syndrome) and severe (Crigler-Najjar syndrome)

19. Pathway Disorders Autosomal recessive disorders
UDP-glucuronyl transferase – mild deficiency (Gilbert syndrome) most common inherited cause of unconjugated hyperbilirubinemia, intermittent jaundice without hemolysis or liver disease, precipitated by dehydration, fasting, menstrual periods, intercurrent illness, trauma, over exertion, nonspecific symptoms such as abdominal cramps, fatigue, and malaise, mild jaundice. Unconjugated hyperbilirubinemia (by definition [bilirubin] < 6 mg/dL, commonly < 3 mg/dL), normal complete blood count, reticulocyte count, and blood smear, normal liver function panel. Treatment is reassurance and avoiding precipitating factors.
UDP-glucuronyl transferase – severe deficiency (Crigler-Najjar syndrome) rare, types 1 and 2 (Arias syndrome). Type 1 almost complete deficiency associated with neonatal unconjugated hyperbilirubinemia (17-50 mg/dL) and kernicterus, hypotonia, deafness, oculomotor palsy, lethargy. Type 2 deficiency unconjugated bilirubin (6-20 mg/dL), persistent jaundice at birth or after. Elevated unconjugated bilirubin with normal liver function panel. Phenobarbital treatment distiguished type 1 (no effect) from type 2 (lowers serum bilirubin 25%). Treatment of bilirubin encephalopathy with plasma exchange transfusion and long-term phototherapy.

20. O2 Binding/Unloading
His64 and His93 either coordinate the Fe or are very close in proximity
Free energy (G) of O2 binding to Hb drives conformational changes → cooperativity
All structures were prepared using Jmol ( and appropriate pdb files (

21. Hemoglobin Structure Changes
Binding of O2 to heme Fe2+ leads to global conformation changes.
2,3-BPG (not show here) binds within the central cavity of deoxyHb, weakly bound to oxyHb (O2 will displace BPG)

22. Factors Affecting Binding of O2
Depends on pH ([H+]), CO2, BPG (DPG), Temp
Bohr effect (two components):
H+ (binds oxyHb to displace O2) BPG (stabilzes deoxyHb)
R: relax, high affinity state T: tense, low affinity state
CO2 binds to N-terminus of subunits (~15% of total CO2)
Carbonic anhydrase catalyzes CO2 + H2O = H2CO3 = HCO3- + H+

23. Integration of Metabolism
SSB
metabolism in brain, nervous tissue and muscle
alcohol processing and effect on metabolic pathways
vitamins in neuromuscular metabolism
ERG
regulation of metabolism and diabetes