2Outline Pyrimidine and purine synthesis Glycogen storage disorders including salvage and degradationGlycogen storage disordersLysosomal storage disordersHeme synthesis and degradationincluding oxygen binding/unloading of hemeIntegration of metabolismincluding lipid synthesis/degradation, glycolysis/gluconeogenesis, TCA cycle and glycogenolysis/glycogen synthesis
3Pyrimidine and Purine Synthesis HCO3- + GlnCPCPSIIR5PPRPPRPKAspOroUMPUMPSIMPGlnGlyCO2AspN10fTHFUTPRNACDPdCDPdUMPDNAGDPADPGln: glutamine CP: carbamoyl phosphate Oro: orotic acid UMP: uridine monophosphateUTP: uridine triphsophate CDP: cytosine diphosphate dCDP: deoxycytosine diphosphatedUMP: deoxyuridine diphosphate dTMP: deoxythymidine monophosphateDNA: deoxyribonucleic acid RNA: ribonucleic acid R5P: ribose-5-phosphatePRPP: 5-phosphoribosylpyrophosphate Gly: glycine N10fTHF: N10-formyltetrahydrofolateAsp: aspartate IMP: inosine monophosphate GDP: guanosine diphosphateADP: adenosine diphosphated dGTP: deoxyguanosine triphosphatedATP: adenosine triphosphateCPSII: cytosolic carbamyolphosphate synthetase, feedback inhibition by UTPUMPS: UMP synthase, a bifunctional enzyme (orotate phosphoribosyltransferase, OMP decarboxylase), deficiency of either activity leads to hereditary orotic aciduriaTS: thymidylate synthase, inhibited by fluorouracilRR: ribonucleotide reductase, inhibited by hydroxyureaFolic acid and its derivatives (for example, THF) are important for biosynthesis and salvage pathways.TSN5,N10-mTHFdTMPdGTPdATPRR
4Pyrimidine and Purine Salvage UTPCDPdCDPdUMPTSN5,N10-mTHFdTMPRNADNAIMPGDPADPdGTPdATPRRUTPRPPUMPTMPRRUTPTAAPTGPRPPHGPTadenosineinosineADAUTP: uridine triphosphate CDP: cytosine diphosphate dCDP: deoxycytosine diphosphatedUMP: deoxyuridine monophosphate dTMP: deoxythymidine monophosphateIMP: inosine monophosphate GDP: guanosine diphosphate ADP: adenosine diphosphatedGTP: deoxyguanosine triphosphate dATP: deoxyadenosine triphosphateUMP: uridine monophosphate TMP: thymidine monophosphate RNA: ribonucleic acidDNA: deoxyribonucleic acid U: uridine T: thymine A: adenine G: guanineN5,N10-mTHF: N5,N10-methylenetetrahydrofolate ADA: adenosine deaminasePNP: purine nucleotide phosphorylase HGPT: hypoxanthine-guanine phosphoribosyl transferaseAPT: adenine phosphoribosyl transferase X: xanthineXO: xanthine oxidase, inhibited by allopurinolThere is feedback inhibition of nucleotide synthesis by productsFolic acid and its derivatives are important for biosynthesis and salvage pathways.HXPNPXXOurateXO
5Pathway 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
6Pathway 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.
7Glycogen Storage Disorders GSGSD0GlycogenG1PG6PGlcUDP-GlcF6PF16BPacid maltaseGSDIIlstransglycosylasebranching enzymeGSDIVhPPGSDVImPPGSDVdebranching enzymeGSDIIIhG6PaseGSDIGSD: glycogen storage disease hPP: hepatic phosphorylase mPP: muscle phosphorylasePPK: phosphorylase kinase hG6Pase: hepatic glucose-6-phosphataseUDP-Glc: uridine diphosphate glucose G1P: glucose-1-phosphate Glc: glucoseF6P: fructose-6-phosphate F16BP: fructose-1,6-bisphosphatePFK-1: phosphofructokinase-1 debranching enzyme: a(1→6)Glcasebranching enzyme: amylo-(1,4 to 1,6) transglycosylaseGSD type 0 GSD type I:von Gierke GSD type II: Pompe GSD type III: CoriGSD type IV: Andersen GSD type V: McArdle GSD type VI: HersGSD type VII: Tarui GSD type IX: GSD type XI: Fanconi-Bickel hepatorenal glycogenosisII, III, V, and VII cause muscle weaknessPFK-1GSDVII
8Pathway 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.
9Pathway DisordersRare autosomal recessive disordersBranching 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.
10Lysosomal Storage Disorders Lipid metabolismLanding, Sandhoff, Tay-Sachs, Krabbe, Gaucher, Niemann Pick (A,B), Wolman, metachromatic leukodystrophy, FabryGlycoprotein metabolismSchindlerMucopolysaccharide metabolismHurler/Scheie, Hunter, Sanfilippo (A,B,C,D), Morquio (A,B), Maroteaux–Lamy, SlyOther lysosomal enzymesPompe, Niemann-Pick (C)The following has a nice review on lysosomal storage diseases:
11Oligosaccharidoses a-Mannosidosis a-MannosidaseGM2 gangliosidosis variant O (Sandhoff-Jatzkewitz disease)b-N-Acetylhexosaminidases A&BAspartylglycosylaminuria4-L-Aspartylglycosylamine amidohydrolase65NANAGalGlcNAc4abbMan65NANAGalGlcNAc43aba72165aManGlcNAcGlcNAcAsnNANAGalGlcNAc43bbab8bManFucosidosisa-Fucosidase65GlcNAcFucb-Mannosidosisb-MannosidaseNANAGalGlcNAc4abMucolipidosis I (Sialidosis)SialidaseGM1 gangliosidosisb-GalactosidaseTypical Asn-GlcNAc OS structureNANA: N-acetylneuraminic acid (sialic acid) Gal: galactoseGlcNAc: N-acetylglucosamine Man: mannose Fuc: fucoseAsn: asparagine OS: oligosaccharide
14General Physical Features Coarse facial features (sometimes with macroglossia)Corneal clouding or related ocular abnormalitiesAngiokeratomaUmbilical/inguinal herniasShort statureDevelopmental delaysJoint or skeletal deformitiesOrganomegaly (especially liver and spleen)Muscle weakness or lack of control (ataxia, seizures, etc.)Neurologic failure/decline or loss of gained developmentThe following has useful information:
15Heme Synthesis mit SCoA + Gly 5AS 5ALA PBG PBGS PBGD HMB FC Heme UPGIIISUPGIIIPPIXPPGOUPGIIIDCCPGIIIMIT: mitochodrion SCoA: succinyl CoA Gly: glycine5ALA: 5-aminolevulinic acid PBG: porphobilinogen5AS: 5-aminolevulinic acid synthase; feedback inhibition by heme and glucose, regulates transcription, mRNA stability, 5AS import; positive regulation by EtOH, sex steroids, barbituates, sulphonamides, anticonvulsantsPBGS: 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: uroporphyrinogenUPGIIIS: 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 poisoningCPGOPPGIX
16Pathway DisordersPBG 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.
17Pathway DisordersCoproporphyrinogen 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.
18Heme Degradation res indirect unconjugated pre-hepatic HO BV Heme BVR BRalbuminalbumin-BRligandinalbuminligandin-BRhepatocyteBR diglucuronideUDP-GTERdirectconjugatedpost-hepaticRES: recticuloendothelial system HO: heme oxidase BV: biliverdinBVR: biliverdin reductase BR: bilirubinUDP-GT: uridinediphosphate-glucuronyl transferase, deficiency mild (Gilbert syndrome) and severe (Crigler-Najjar syndrome)
19Pathway 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.
20O2 Binding/UnloadingHis64 and His93 either coordinate the Fe or are very close in proximityFree energy (G) of O2 binding to Hb drives conformational changes → cooperativityAll structures were prepared using Jmol (http://jmol.sourceforge.net/) and appropriate pdb files (http://www.pdb.org/pdb/home/home.do).
21Hemoglobin 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)
22Factors Affecting Binding of O2 Depends on pH ([H+]), CO2, BPG (DPG), TempBohr effect (two components):H+ (binds oxyHb to displace O2) BPG (stabilzes deoxyHb)R: relax, high affinity state T: tense, low affinity stateCO2 binds to N-terminus of subunits (~15% of total CO2)Carbonic anhydrase catalyzes CO2 + H2O = H2CO3 = HCO3- + H+
23Integration of Metabolism SSBmetabolism in brain, nervous tissue and musclealcohol processing and effect on metabolic pathwaysvitamins in neuromuscular metabolismERGregulation of metabolism and diabetes