enzymology (3)

8/9/2019 ENZYMOLOGY (3)

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MORE ON ENZYMESMORE ON ENZYMES

JULIUS P. MARIO, RMT, MSJULIUS P. MARIO, RMT, MS


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ENZYME REGULATIONENZYME REGULATION

Feedback ControlFeedback Control

ProenzymesProenzymes

AllosterismAllosterism

Protein ModificationProtein Modification

IsoenzymesIsoenzymes


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FEEDBACK CONTROLFEEDBACK CONTROL

Formation of the product inhibits anFormation of the product inhibits anearlier reaction in a sequenceearlier reaction in a sequence

In the reaction,In the reaction, E1 E2 E3E1 E2 E3

AA BB CC DD

D may inhibit E1 activity by competitive orD may inhibit E1 activity by competitive ornoncompetitive inhibitionnoncompetitive inhibition

When D is low, all three reactions proceed rapidlyWhen D is low, all three reactions proceed rapidly

When D is high, E1 becomes inhibitedWhen D is high, E1 becomes inhibited


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PROENZYMESPROENZYMES Inactive form of an enzymeInactive form of an enzyme

Also known as zymogenAlso known as zymogenActivated by cleavage of excess polypeptideActivated by cleavage of excess polypeptide

chainchain

Cleavage of peptide promotes structuralCleavage of peptide promotes structuralchanges, thus, functionalchanges, thus, functional

Examples areExamples are TrypsinogenTrypsinogen = cleavage of the 6 amino acids= cleavage of the 6 amino acids

from the Nfrom the N--terminal by enteropeptidase converts itterminal by enteropeptidase converts itto activeto active trypsintrypsin

AngiotensinogenAngiotensinogen = cleavage of the last 2 amino= cleavage of the last 2 aminoacids from the Cacids from the C--terminal byterminal byACEACE converts it to aconverts it to a

vasoactive octapeptide,vasoactive octapeptide, angiotensinangiotensin


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PROENZYMESPROENZYMES

ChymotrypsinogenChymotrypsinogen = cleavage of the 245= cleavage of the 245residues long polypeptide between Rresidues long polypeptide between R--15 and15 andII--16 from the N16 from the N--terminal by trypsin converts itterminal by trypsin converts itto activeto active --chymotrypsin but the fully activechymotrypsin but the fully active

form isform is --chymotrypsinchymotrypsin


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PROENZYMESPROENZYMES

Some enzymes have proenzymes becauseSome enzymes have proenzymes becausethey are highly destructive when producedthey are highly destructive when produced

directly in their active formsdirectly in their active forms

Should these leak or exist in excess,Should these leak or exist in excess,inhibitory proteins bind them to renderinhibitory proteins bind them to renderthem inactivethem inactive


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ALLOSTERISMALLOSTERISM

If a substance binds noncovalently andIf a substance binds noncovalently andreversibly to a sitereversibly to a site other than the activeother than the active

site,site, It may inhibit the enzymeIt may inhibit the enzyme

(Negative modulation)(Negative modulation)

Itmay stimulate enzyme actionItmaystimulate enzyme action

(Positive modulation)(Positive modulation)


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ALLOSTERISMALLOSTERISM

AlloAllomeans other;means other; stericstericmeans shapemeans shape

Possible conformationalchanges affect thePossible conformationalchanges affect thebehavior of proteinsbehavior of proteins

Due tomultiple forms of theDue tomultiple forms of the quaternaryquaternarystructurestructure of some allosteric enzymesof some allosteric enzymes

A substance that modifies 4A substance that modifies 4o structure andstructure and

thus, affect behavior is anthus, affect behavior is an allosteric effectorallosteric effector AllostericeffectorsAllostericeffectors are substrates,are substrates,

activators, and inhibitorsactivators, and inhibitors


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ALLOSTERIC PROTEINSALLOSTERIC PROTEINS Proteins in which subtle changes at one siteProteins in which subtle changes at one site

affects theaffects the structure and functionstructure and function ofofanother siteanother site

Due toDue to cooperativecooperative effectseffects

Depicted asDepicted as sigmoidal curvesigmoidal curve on enzymeon enzyme

kineticskinetics

Examples areExamples are Aspartate transcarbamoylaseAspartate transcarbamoylase(ATCase) &(ATCase) & HemoglobinHemoglobin


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NONALLOSTERIC PROTEINSNONALLOSTERIC PROTEINS

At first, dependent on the [S] with aAt first, dependent on the [S] with amaximal rate not dependent on [S]maximal rate not dependent on [S]

anymoreanymore

Depicted as aDepicted as a hyperbolichyperbolic curve oncurve onMichaelisMichaelis--Menten kineticsMenten kinetics

Examples areExamples are chymotrypsinchymotrypsin && myoglobinmyoglobin


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HOMOTROPIC vs. HETEROTROPICHOMOTROPIC vs. HETEROTROPIC

EffectsEffects HomotropicHomotropic effectseffects areare allostericallosteric

interactionsinteractions thatthat occuroccur whenwhen severalseveralidenticalidentical moleculesmolecules areare boundbound toto aaproteinprotein..

HeterotropicHeterotropic effectseffects areare allostericallostericinteractionsinteractions thatthat occuroccur whenwhen differentdifferentsubstancessubstancesareare boundbound toto aa proteinprotein..


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PROTEIN MODIFICATIONPROTEIN MODIFICATION Modification is usually a change in theModification is usually a change in the

primary structure,primary structure, typically by the additiontypically by the additionof a functional group covalently bound toof a functional group covalently bound tothe apoenzymethe apoenzyme

BestBest--known example is theknown example is theactivation/inhibition ofphosphorylationactivation/inhibition ofphosphorylation Glycogen phosphorylaseGlycogen phosphorylase is active when it isis active when it is

phosphorylated at its serine or tyrosinephosphorylated at its serine or tyrosineresidueresidue

Pyruvate kinasePyruvate kinase from the liver is inactivefrom the liver is inactive

when it is phosphorylatedwhen it is phosphorylated


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ISOENZYMESISOENZYMES Enzymes that perform the same functionEnzymes that perform the same function

but have different combinations ofbut have different combinations ofsubunits, thus have different 4subunits, thus have different 4o structuresstructures

Have different electrophoretic mobilities,Have different electrophoretic mobilities,Km, and originsKm, and origins

Act on the same substrateAct on the same substrate

LDH has 4 subunitsLDH has 4 subunits LD1 (LD1 (HH44), LD2 (), LD2 (HH33MM), LD3 (), LD3 (HH22MM22), LD4), LD4

((H

MH

M33) & LD5 () & LD5 (MM44))


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CARBOXYPEPTIDASESCARBOXYPEPTIDASES

CleaveCleave peptidespeptides andand proteinsproteins atat thethe carboxylcarboxylterminalterminal ofof aa particularparticular aminoamino acidacid inin thethechainchain

TrypsinTrypsin == atatlysyllysyloror arginylarginyl

ChymotrypsinChymotrypsin == atatphenylalanylphenylalanyl,,

tryptophanyltryptophanyl oror tyrosyltyrosylasaswellwellasas L,L, HH && QQ

PepsinPepsin == samesame withwith chymotrypsinchymotrypsin andand othersothers

ThermolysinThermolysin == atatisoleucylisoleucyl,, leucylleucyl oror valylvalyl


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PROTEASE FAMILYPROTEASE FAMILY

All members have similar chemical formAll members have similar chemical form

Serine ProteasesSerine Proteases = cleaves at seryl residues= cleaves at seryl residues((chymotrypsinchymotrypsin,, trypsintrypsinandand elastinelastin))

Cysteine proteaseCysteine protease = cleaves at cysteinyl= cleaves at cysteinylresidue (residue (papainpapain, a meat tenderizer), a meat tenderizer)

Aspartyl proteaseAspartyl protease = a pair of aspartate side= a pair of aspartate sidechains, sometimes on different subunitschains, sometimes on different subunitsparticipate in the reaction (participate in the reaction (pepsinpepsin andand HIVHIVproteaseprotease))


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ABZYMESABZYMESAntibodies elicited by antigenic proteinsAntibodies elicited by antigenic proteins

Designer enzymes which can catalyze aDesigner enzymes which can catalyze awide variety of reactionswide variety of reactions

Usually areUsually are transitiontransition--state analogsstate analogs whichwhichwhen introduced into the body becomeswhen introduced into the body becomesimmunogenicimmunogenic

NN--(5(5--phosphopyridoxyl)phosphopyridoxyl)--LL--lysinelysine isisthe transitionthe transition--state analog for the reactionstate analog for the reactionbetween an amino acid and pyridoxalbetween an amino acid and pyridoxal--55--phosphatephosphate


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CELLULAR LEVELCELLULAR LEVEL SER enzymesSER enzymes

Detoxification by theDetoxification by the cytochrome Pcytochrome P--450450enzymesenzymes

Lipid synthesis & degradation byLipid synthesis & degradation byCytosolic sideCytosolic side = cyt b5, cyt b5reductase,= cyt b5, cyt b5reductase,NADPHNADPH--ferrihemoproteinreductase,ATPase,5ferrihemoproteinreductase,ATPase,5--NT, glycolipid mannosyl transferases,NT, glycolipid mannosyl transferases,hydroxymethylglutarylhydroxymethylglutaryl--CoAreductase, some 30CoAreductase, some 30

enzymes forsteroid synthesis and 20 enzymesenzymes forsteroid synthesis and 20 enzymesoflipid synthesisoflipid synthesis

Luminal sideLuminal side = cyt P= cyt P--450, cyt P450, cyt P--450reductase,450reductase,glucoseglucose--66--phosphatase andphosphatase and --glucuronidaseglucuronidase


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RERRER Transport enzymes for glycosylation of newlyTransport enzymes for glycosylation of newly

formed proteins;formed proteins; flippasesflippases for phospholipidfor phospholipidsynthesissynthesis

GOLGI BODYGOLGI BODY Enzymes for posttranslational modifications ofEnzymes for posttranslational modifications of

proteins synthesized on membranes as well asproteins synthesized on membranes as well asrecycling of membrane material;recycling of membrane material; 55--NT,NT,NADH:cytcoxidoreductase,NADPH:cytcNADH:cytcoxidoreductase,NADPH:cytcreductase, UDPreductase, UDP--galactosegalactose--NN--acetylglucosamineacetylglucosamine----DD--galactosyltransferase,galactosyltransferase,andand manymanyglycosylationenzymesglycosylationenzymes


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LYSOSOMESLYSOSOMES

Hydrolytic enzymes such asHydrolytic enzymes such as proteinases,proteinases,glycosidases,lipases,phosphatases,glycosidases,lipases,phosphatases,nucleases,nucleases,andand sulfatasessulfatases

MICROBODIESMICROBODIES

MainlyMainly oxidative enzymesoxidative enzymes

VACUOLESVACUOLES

VV--type Htype H++--translocatingATPasetranslocatingATPase


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BRUSH BORDER ENZYMESBRUSH BORDER ENZYMES

Disaccharidases found on the microvilli ofDisaccharidases found on the microvilli ofthe small intestinethe small intestine

Once lost, can be reOnce lost, can be re--synthesized viasynthesized viaenzyme inductionenzyme induction

Substrates commonly acted upon areSubstrates commonly acted upon aremaltose, sucrose, and lactosemaltose, sucrose, and lactose


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ENZYMES AS VIRULENCEENZYMES AS VIRULENCEFACTORSFACTORS

HelicobacterpyloriHelicobacterpylorisurvives the harshsurvives the harshgastricmilieu bythegastricmilieu bythe ureaseurease on its cellon its cellwall. The ammoniaproduced counters thewall. The ammoniaproduced counters theharmfuleffects ofthe acid.harmfuleffects ofthe acid.

Some pathogenic cocci are capable ofSome pathogenic cocci are capable ofproducingproducing hemolysinshemolysins which interferewhich interferewith oxygen transport in an organismwith oxygen transport in an organism


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EFFECTS ON ENZYMESEFFECTS ON ENZYMES Bactericidal antibiotics for both GramBactericidal antibiotics for both Gram

positive and Gram negative organisms actpositive and Gram negative organisms actvia inhibition of cell wall synthesizingvia inhibition of cell wall synthesizing

proteinsynthaseproteinsynthase (e.g. Carbapenems)(e.g. Carbapenems)

Binds to theBinds to the subunit ofsubunit ofRNARNA

polymerasepolymerase to inhibit transcription ofto inhibit transcription ofmostly Gram+ bacteria & Mycobacteriamostly Gram+ bacteria & Mycobacteria(e.g. Rifampin)(e.g. Rifampin)


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An example of a medicinal enzyme inhibitor isAn example of a medicinal enzyme inhibitor issildenafil (Viagra), a common treatment for malesildenafil (Viagra), a common treatment for male

erectile dysfunction.erectile dysfunction.

This compound is a potent inhibitor of cGMPThis compound is a potent inhibitor of cGMPspecific phosphodiesterase type 5, the enzymespecific phosphodiesterase type 5, the enzymethat degrades thethat degrades the signallingsignalling moleculemolecule cycliccyclic

guanosine monophosphateguanosine monophosphate..

This signalling molecule triggers smooth muscleThis signalling molecule triggers smooth musclerelaxation and allows blood flow into therelaxation and allows blood flow into the corpuscorpus

cavernosumcavernosum, which causes an erection. Since, which causes an erection. Sincethe drug decreases the activity of the enzymethe drug decreases the activity of the enzymethat halts the signal, it makes this signal last forthat halts the signal, it makes this signal last fora longer period of time.a longer period of time.


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Another example of the structural similarity ofAnother example of the structural similarity ofsome inhibitors to the substrates of the enzymessome inhibitors to the substrates of the enzymes

they target is seen in the figure comparing thethey target is seen in the figure comparing thedrugdrug methotrexatemethotrexate toto folic acidfolic acid. Folic acid is the. Folic acid is theoxidised form of the substrate ofoxidised form of the substrate ofdihydrofolatedihydrofolatereductasereductase, an enzyme that is potently inhibited, an enzyme that is potently inhibited

by methotrexate. Methotrexate blocks the actionby methotrexate. Methotrexate blocks the actionof dihydrofolate reductase and thereby haltsof dihydrofolate reductase and thereby haltsthymidinethymidine biosynthesis. This block ofbiosynthesis. This block ofnucleotidenucleotidebiosynthesis is selectively toxic to rapidlybiosynthesis is selectively toxic to rapidly

growing cells, therefore methotrexate is oftengrowing cells, therefore methotrexate is oftenused in cancerused in cancer chemotherapychemotherapy..


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Drugs also are used to inhibit enzymesDrugs also are used to inhibit enzymes

needed for the survival ofneeded for the survival ofpathogenspathogens. For. Forexample, bacteria are surrounded by aexample, bacteria are surrounded by athickthick cell wallcell wall made of a netmade of a net--like polymerlike polymercalledcalled peptidoglycanpeptidoglycan. Many antibiotics. Many antibiotics

such assuch as penicillinpenicillin andand vancomycinvancomycin inhibitinhibitthe enzymes (thethe enzymes (the transpeptidasetranspeptidase from thefrom thebacteriabacteria StreptomycesStreptomyces R61) that produceR61) that produce

and then crossand then cross--link the strands of thislink the strands of thispolymer together. This causes the cell wallpolymer together. This causes the cell wallto lose strength and the bacteria to burst.to lose strength and the bacteria to burst.


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Drug designDrug design is facilitated when an enzymeis facilitated when an enzymethat is essential to the pathogen's survivalthat is essential to the pathogen's survivalis absent or very different in humans.is absent or very different in humans.

Humans do not make peptidoglycan,Humans do not make peptidoglycan,

therefore inhibitors of this process aretherefore inhibitors of this process areselectively toxic to bacteria.selectively toxic to bacteria.

Selective toxicity is also produced inSelective toxicity is also produced inantibiotics by exploiting differences in theantibiotics by exploiting differences in thestructure of thestructure of the ribosomesribosomes in bacteria, orin bacteria, orhow they makehow they make fatty acidsfatty acids


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AcCHE, an enzyme found in animals from insects toAcCHE, an enzyme found in animals from insects tohumans. It is essential to nerve cell function through itshumans. It is essential to nerve cell function through itsmechanism of breaking down the neurotransmittermechanism of breaking down the neurotransmitter

acetylcholineacetylcholine into its constituents,into its constituents, acetateacetate andand cholinecholine..

This is somewhat unique among neurotransmitters asThis is somewhat unique among neurotransmitters asmost, includingmost, including serotoninserotonin,, dopaminedopamine, and, andnorepinephrinenorepinephrine, are absorbed from the, are absorbed from the synaptic cleftsynaptic cleft

rather than cleaved.rather than cleaved.

Reversible competitive inhibitorsReversible competitive inhibitors, such as, such asedrophoniumedrophonium,, physostigminephysostigmine, and, and neostigmineneostigmine, are used, are usedin the treatment ofin the treatment ofmyasthenia gravismyasthenia gravis and inand inanaesthesia. Theanaesthesia. The carbamatecarbamate pesticides are alsopesticides are alsoexamples of reversible AChE inhibitors.examples of reversible AChE inhibitors.

TheThe organophosphateorganophosphate insecticides such asinsecticides such as malathionmalathion,,parathionparathion, and, and chlorpyrifoschlorpyrifos irreversibly inhibitirreversibly inhibitacetylcholinesterase.acetylcholinesterase.


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ENZYME DEFICIENCIESENZYME DEFICIENCIES


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MUCOPOLYSACCHARIDOSISMUCOPOLYSACCHARIDOSISMPS Type Eponym Deficient EnzymeMPS Type Eponym Deficient Enzyme

I H Hurler aI H Hurler a--iduronidaseiduronidase

I H/S HurlerI H/S Hurler--Scheie aScheie a--iduronidaseiduronidase

I S Scheie aI S Scheie a--iduronidaseiduronidase

II Hunter Iduronidase sulfataseII Hunter Iduronidase sulfatase

III Sanfilippo A Heparan sulfataseIII Sanfilippo A Heparan sulfatase

Sanfilippo BSanfilippo B NN--acetylglucosaminidaseacetylglucosaminidase

Sanfilippo C Acetyl CoA glucosamineSanfilippo C Acetyl CoA glucosamineacetyltransferaseacetyltransferase

Sanfilippo DSanfilippo D NN--acetylglucosamineacetylglucosamine--66--sulfatasesulfatase

IV Morquio A GalactosamineIV Morquio A Galactosamine--66--sulfatasesulfatase

Morquio BMorquio B bb--galactosidasegalactosidaseV NonexistentV Nonexistent

VI Maroteaux VI Maroteaux--LamyLamy NN--acetylhexosamineacetylhexosamine--44--sulfatasesulfatase

VII Sly VII Sly bb--glucuronidaseglucuronidase

IX Hyaluronidase HyaluronidaseIX Hyaluronidase Hyaluronidase

DeficiencyDeficiency


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GLYCOGEN STORAGE DISEASESGLYCOGEN STORAGE DISEASES

TypeType -- Enzyme DeficientEnzyme Deficient --Clinical FeaturesClinical FeaturesII ((von Gierkevon Gierke)) -- Liver and kidneyLiver and kidney GlucoseGlucose--66--phosphatasephosphatase

Hepatomegaly, lactic acidosis, hyperlipidemia, severe fastingHepatomegaly, lactic acidosis, hyperlipidemia, severe fastinghypoglycemiahypoglycemia

II (Pompe)II (Pompe)--All tissuesAll tissuesalpha 1,4alpha 1,4--glucosidaseglucosidaseCardiomegaly, muscle weakness, death in infancy and adultsCardiomegaly, muscle weakness, death in infancy and adults

III (CoriIII (Cori--Forbes)Forbes)--All tissuesAll tissuesdebrancher enzymedebrancher enzymeHepatomgaly, muscle weakness, fasting hypoglycermiaHepatomgaly, muscle weakness, fasting hypoglycermia

IV (Andersen)IV (Andersen)-- All tissuesAll tissuesbrancher enzymebrancher enzyme

Portal cirrhosis, death in infancyPortal cirrhosis, death in infancy

V (McArdle)V (McArdle)-- Muscle phosphorylaseMuscle phosphorylase

Pain and stiffness after exertion; myoglobinuriaPain and stiffness after exertion; myoglobinuria


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VI (Hers)VI (Hers) Liver phosphorylaseLiver phosphorylase Hepatomegaly,Hepatomegaly,mild fastingmild fasting

hypoglycemiahypoglycemia

VII (Tarui)VII (Tarui) Muscle and liverMuscle and liverphosphofructokinasephosphofructokinase

Pain and stiffnessPain and stiffnesson exertionon exertion

VIIIVIII Brain and liverBrain and liveradenylate kinaseadenylate kinase

Spasticity,Spasticity,decerebration,decerebration,high urinaryhigh urinary

catecholamines,catecholamines,death in infancydeath in infancy

IXIX Liver phosphorylaseLiver phosphorylasekinasekinase

Hepatomegaly,Hepatomegaly,occasional fastingoccasional fasting

hypoglycemiahypoglycemia

XX Liver and muscle cAMPLiver and muscle cAMP--

dependent kinasedependent kinase

HepatomegalyHepatomegaly

onlyonly


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LIPIDOSESLIPIDOSES

a group of inherited disordersa group of inherited disorderscharacterized by thecharacterized by the

accumulation of lipids inaccumulation of lipids intissuestissues especially the brainespecially the brain

due todue to

deficiency in a particulardeficiency in a particularsphingolipid catabolic enzymesphingolipid catabolic enzyme


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NiemannNiemann--Pick diseasePick disease Deficiency inDeficiency in sphingomyelinasesphingomyelinase andand

accumulation of sphingomyelinaccumulation of sphingomyelin

Gauchers diseaseGauchers disease

Deficiency inDeficiency in FF--DD--glucosidaseglucosidase andand

accumulation of glucocerebrosideaccumulation of glucocerebroside


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Krabbes diseaseKrabbes disease Deficiency inDeficiency in FF--DD--galactosidasegalactosidase andand

accumulation ofaccumulation of galactocerebsidesgalactocerebsides

Fabrys diseaseFabrys disease

Deficiency inDeficiency in EE--DD--galactosidasegalactosidase andand

accumulation of ceramide trihexosideaccumulation of ceramide trihexoside


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TayTay--Sachs diseaseSachs disease Deficiency inDeficiency in FF--DD--hexaminidase Ahexaminidase A

andand accumulation of ganglioside GMaccumulation of ganglioside GM22

Metachromatic LeukodystrophyMetachromatic Leukodystrophy

Deficiency inDeficiency in sulfatide sulfatasesulfatide sulfatase andandaccumulation ofaccumulation of FF--sulfogalactocerebrosidesulfogalactocerebroside

enzymology (3)

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