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

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

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

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

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

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.

Glycogen Storage Disorders GS 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 http://emedicine.medscape.com/article/1116574-overview http://themedicalbiochemistrypage.org/glycogen.php#storage 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

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.

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.

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: http://www.sciencedirect.com/science/article/pii/S0925443902002296

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

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

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

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: http://www.lysosomallearning.com/healthcare/about/lsd_hc_abt_lsd.asp

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

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.

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.

Heme Degradation res indirect unconjugated pre-hepatic HO BV Heme BVR BR 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)

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.

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 (http://jmol.sourceforge.net/) and appropriate pdb files (http://www.pdb.org/pdb/home/home.do).

Hemoglobin Structure Changes http://en.wikipedia.org/wiki/Hemoglobin 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)

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+ http://en.wikipedia.org/wiki/Bohr_Effect

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