MORE ON ENZYMESMORE ON ENZYMES

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

ENZYME REGULATIONENZYME REGULATION

Feedback ControlFeedback ControlProenzymesProenzymesAllosterismAllosterismProtein ModificationProtein ModificationIsoenzymesIsoenzymes

FEEDBACK CONTROLFEEDBACK CONTROL

Formation of the product inhibits an Formation of the product inhibits an earlier reaction in a sequenceearlier reaction in a sequence

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

A A B B C C D D D may inhibit E1 activity by competitive or D may inhibit E1 activity by competitive or

noncompetitive inhibitionnoncompetitive inhibition When D is low, all three reactions proceed When D is low, all three reactions proceed

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

PROENZYMESPROENZYMES Inactive form of an enzymeInactive form of an enzymeAlso known as zymogenAlso known as zymogenActivated by cleavage of excess Activated by cleavage of excess

polypeptide chain polypeptide chain Cleavage of peptide promotes structural Cleavage of peptide promotes structural

changes, thus, functionalchanges, thus, functionalExamples areExamples are

TrypsinogenTrypsinogen = cleavage of the 6 amino acids = cleavage of the 6 amino acids from the N-terminal by enteropeptidase from the N-terminal by enteropeptidase converts it to active converts it to active trypsintrypsin

AngiotensinogenAngiotensinogen = cleavage of the last 2 = cleavage of the last 2 amino acids from the C-terminal by amino acids from the C-terminal by ACEACE converts it to a vasoactive octapeptide, converts it to a vasoactive octapeptide, angiotensinangiotensin

PROENZYMESPROENZYMESChymotrypsinogen Chymotrypsinogen = cleavage of the = cleavage of the

245 residues long polypeptide between 245 residues long polypeptide between R-15 and I-16 from the N-terminal by R-15 and I-16 from the N-terminal by trypsin converts it to active trypsin converts it to active пп--chymotrypsin but the fully active form is chymotrypsin but the fully active form is αα-chymotrypsin-chymotrypsin

PROENZYMESPROENZYMES

Some enzymes have proenzymes Some enzymes have proenzymes because they are highly destructive because they are highly destructive when produced directly in their when produced directly in their active formsactive forms

Should these leak or exist in excess, Should these leak or exist in excess, inhibitory proteins bind them to inhibitory proteins bind them to render them inactiverender them inactive

ALLOSTERISMALLOSTERISM

If a substance binds noncovalently If a substance binds noncovalently and reversibly to a site and reversibly to a site other than other than the active site,the active site,It may inhibit the enzyme It may inhibit the enzyme

(Negative modulation)(Negative modulation)It may stimulate enzyme actionIt may stimulate enzyme action

(Positive modulation)(Positive modulation)

ALLOSTERISMALLOSTERISMAllo Allo means other; means other; stericsteric means shape means shapePossible conformational changes affect Possible conformational changes affect

the behavior of proteinsthe behavior of proteinsDue to multiple forms of the Due to multiple forms of the quaternary quaternary

structurestructure of some allosteric enzymes of some allosteric enzymesA substance that modifies 4A substance that modifies 4o structure structure

and thus, affect behavior is an and thus, affect behavior is an allosteric effectorallosteric effector

Allosteric effectors Allosteric effectors are substrates, are substrates, activators, and inhibitorsactivators, and inhibitors

ALLOSTERIC PROTEINSALLOSTERIC PROTEINSProteins in which subtle changes at Proteins in which subtle changes at

one site affects the one site affects the structure and structure and functionfunction of another site of another site

Due to Due to cooperativecooperative effects effects

Depicted as Depicted as sigmoidal curvesigmoidal curve on on enzyme kineticsenzyme kinetics

Examples are Examples are Aspartate Aspartate transcarbamoylasetranscarbamoylase (ATCase) & (ATCase) & HemoglobinHemoglobin

NONALLOSTERIC NONALLOSTERIC PROTEINSPROTEINS

At first, dependent on the [S] with a At first, dependent on the [S] with a maximal rate not dependent on [S] maximal rate not dependent on [S] anymoreanymore

Depicted as a Depicted as a hyperbolichyperbolic curve on curve on Michaelis-Menten kineticsMichaelis-Menten kinetics

Examples are Examples are chymotrypsin chymotrypsin & & myoglobinmyoglobin

HOMOTROPIC vs. HOMOTROPIC vs. HETEROTROPICHETEROTROPIC

EffectsEffectsHomotropic effects are allosteric Homotropic effects are allosteric

interactions that occur when several interactions that occur when several identical moleculesidentical molecules are bound to a are bound to a protein.protein.

Heterotropic effects are allosteric Heterotropic effects are allosteric interactions that occur when interactions that occur when different substancesdifferent substances are bound to are bound to a protein. a protein.

PROTEIN MODIFICATIONPROTEIN MODIFICATIONModification is usually a change in the Modification is usually a change in the

primary structure, primary structure, typically by the typically by the addition of a functional group addition of a functional group covalently bound to the apoenzymecovalently bound to the apoenzyme

Best-known example is the Best-known example is the activation/inhibition of activation/inhibition of phosphorylationphosphorylationGlycogen phosphorylaseGlycogen phosphorylase is active when it is active when it

is phosphorylated at its serine or tyrosine is phosphorylated at its serine or tyrosine residueresidue

Pyruvate kinasePyruvate kinase from the liver is inactive from the liver is inactive when it is phosphorylatedwhen it is phosphorylated

ISOENZYMESISOENZYMESEnzymes that perform the same Enzymes that perform the same

function but have different function but have different combinations of subunits, thus have combinations of subunits, thus have different 4different 4o structures structures

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

Act on the same substrateAct on the same substrate

LDH has 4 subunitsLDH has 4 subunitsLD1 (LD1 (HH44), LD2 (), LD2 (HH33MM), LD3 (), LD3 (HH22MM22), LD4 ), LD4

((HMHM33) & LD5 () & LD5 (MM44))

CARBOXYPEPTIDASESCARBOXYPEPTIDASESCleave peptides and proteins at the Cleave peptides and proteins at the

carboxyl terminal of a particular amino carboxyl terminal of a particular amino acid in the chainacid in the chain

TrypsinTrypsin = at = at lysyllysyl or or arginyl arginyl ChymotrypsinChymotrypsin = at = at phenylalanylphenylalanyl, ,

tryptophanyl tryptophanyl or or tyrosyltyrosyl as well as well as as L, HL, H & & QQ

PepsinPepsin = same with chymotrypsin and = same with chymotrypsin and othersothers

ThermolysinThermolysin = at = at isoleucylisoleucyl, , leucyl leucyl or or valylvalyl

PROTEASE FAMILYPROTEASE FAMILY All members have similar chemical formAll members have similar chemical form

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

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

Aspartyl proteaseAspartyl protease = a pair of aspartate = a pair of aspartate side chains, sometimes on different side chains, sometimes on different subunits participate in the reaction (subunits participate in the reaction (pepsinpepsin and and HIV proteaseHIV protease))

ABZYMESABZYMESAntibodies elicited by antigenic proteinsAntibodies elicited by antigenic proteinsDesigner enzymes which can catalyze a Designer enzymes which can catalyze a

wide variety of reactionswide variety of reactionsUsually are Usually are transition-state analogs transition-state analogs

which when introduced into the body which when introduced into the body becomes immunogenicbecomes immunogenic

NNαα-(5’-phosphopyridoxyl)-L-lysine-(5’-phosphopyridoxyl)-L-lysine is is the transition-state analog for the the transition-state analog for the reaction between an amino acid and reaction between an amino acid and pyridoxal-5’-phosphatepyridoxal-5’-phosphate

CELLULAR LEVELCELLULAR LEVELSER enzymesSER enzymes

Detoxification by the Detoxification by the cytochrome P-450 cytochrome P-450 enzymesenzymes

Lipid synthesis & degradation by Lipid synthesis & degradation by Cytosolic sideCytosolic side = cyt b5, cyt b5 reductase, = cyt b5, cyt b5 reductase,

NADPH-ferrihemoprotein reductase, NADPH-ferrihemoprotein reductase, ATPase, 5’-NT, glycolipid mannosyl ATPase, 5’-NT, glycolipid mannosyl transferases, hydroxymethylglutaryl-CoA transferases, hydroxymethylglutaryl-CoA reductase, some 30 enzymes for steroid reductase, some 30 enzymes for steroid synthesis and 20 enzymes of lipid synthesissynthesis and 20 enzymes of lipid synthesis

Luminal sideLuminal side = cyt P-450, cyt P-450 = cyt P-450, cyt P-450 reductase, glucose-6-phosphatase and reductase, glucose-6-phosphatase and ββ--glucuronidaseglucuronidase

RERRERTransport enzymes for glycosylation of Transport enzymes for glycosylation of

newly formed proteins; newly formed proteins; flippasesflippases for for phospholipid synthesis phospholipid synthesis

GOLGI BODYGOLGI BODYEnzymes for posttranslational modifications Enzymes for posttranslational modifications

of proteins synthesized on membranes as of proteins synthesized on membranes as well as recycling of membrane material; well as recycling of membrane material; 5’-5’-NT, NADH:cyt c oxidoreductase, NT, NADH:cyt c oxidoreductase, NADPH:cyt c reductase, UDP-NADPH:cyt c reductase, UDP-galactose-N-acetylglucosamine-galactose-N-acetylglucosamine-ββ-D--D-galactosyltransferase, galactosyltransferase, and and many many glycosylation enzymesglycosylation enzymes

LYSOSOMESLYSOSOMESHydrolytic enzymes such as Hydrolytic enzymes such as

proteinases, glycosidases, lipases, proteinases, glycosidases, lipases, phosphatases, nucleases, phosphatases, nucleases, and and sulfatasessulfatases

MICROBODIESMICROBODIESMainly Mainly oxidative enzymesoxidative enzymes

VACUOLESVACUOLESV-type HV-type H++-translocating ATPase-translocating ATPase

BRUSH BORDER ENZYMESBRUSH BORDER ENZYMESDisaccharidases found on the Disaccharidases found on the

microvilli of the small intestinemicrovilli of the small intestine

Once lost, can be re-synthesized via Once lost, can be re-synthesized via enzyme inductionenzyme induction

Substrates commonly acted upon are Substrates commonly acted upon are maltose, sucrose, and lactosemaltose, sucrose, and lactose

ENZYMES AS VIRULENCE ENZYMES AS VIRULENCE FACTORSFACTORS

Helicobacter pyloriHelicobacter pylori survives the harsh survives the harsh gastric milieu by the gastric milieu by the urease urease on its cell on its cell wall. The ammonia produced counters wall. The ammonia produced counters the harmful effects of the acid.the harmful effects of the acid.

Some pathogenic cocci are capable of Some pathogenic cocci are capable of producing producing hemolysins hemolysins which interfere which interfere with oxygen transport in an organismwith oxygen transport in an organism

EFFECTS ON ENZYMESEFFECTS ON ENZYMESBactericidal antibiotics for both Gram Bactericidal antibiotics for both Gram

positive and Gram negative positive and Gram negative organisms act via inhibition of cell organisms act via inhibition of cell wall synthesizing wall synthesizing protein synthaseprotein synthase (e.g. Carbapenems)(e.g. Carbapenems)

Binds to the β subunit of Binds to the β subunit of RNA RNA polymerasepolymerase to inhibit transcription to inhibit transcription of mostly Gram+ bacteria & of mostly Gram+ bacteria & Mycobacteria (e.g. Rifampin)Mycobacteria (e.g. Rifampin)

An example of a medicinal enzyme inhibitor An example of a medicinal enzyme inhibitor is sildenafil (Viagra), a common treatment is sildenafil (Viagra), a common treatment for male erectile dysfunction.for male erectile dysfunction.

This compound is a potent inhibitor of This compound is a potent inhibitor of cGMP specific phosphodiesterase type 5, cGMP specific phosphodiesterase type 5, the enzyme that degrades the the enzyme that degrades the signallingsignalling molecule molecule cyclic cyclic guanosineguanosine monophosphatemonophosphate..

This signalling molecule triggers smooth This signalling molecule triggers smooth muscle relaxation and allows blood flow into muscle relaxation and allows blood flow into the the corpus corpus cavernosumcavernosum, which causes an , which causes an erection. Since the drug decreases the erection. Since the drug decreases the activity of the enzyme that halts the signal, activity of the enzyme that halts the signal, it makes this signal last for a longer period it makes this signal last for a longer period of time. of time.

Another example of the structural similarity Another example of the structural similarity of some inhibitors to the substrates of the of some inhibitors to the substrates of the enzymes they target is seen in the figure enzymes they target is seen in the figure comparing the drug comparing the drug methotrexatemethotrexate to to folic acidfolic acid. Folic acid is the oxidised form of . Folic acid is the oxidised form of the substrate of the substrate of dihydrofolatedihydrofolate reductasereductase, an , an enzyme that is potently inhibited by enzyme that is potently inhibited by methotrexate. Methotrexate blocks the methotrexate. Methotrexate blocks the action of dihydrofolate reductase and action of dihydrofolate reductase and thereby halts thereby halts thymidinethymidine biosynthesis. This biosynthesis. This block of block of nucleotidenucleotide biosynthesis is biosynthesis is selectively toxic to rapidly growing cells, selectively toxic to rapidly growing cells, therefore methotrexate is often used in therefore methotrexate is often used in cancer cancer chemotherapychemotherapy. .

Drugs also are used to inhibit enzymes Drugs also are used to inhibit enzymes needed for the survival of needed for the survival of pathogenspathogens. For . For example, bacteria are surrounded by a example, bacteria are surrounded by a thick thick cell wallcell wall made of a net-like polymer made of a net-like polymer called called peptidoglycanpeptidoglycan. Many antibiotics . Many antibiotics such as such as penicillinpenicillin and and vancomycinvancomycin inhibit inhibit the enzymes (the transpeptidase from the enzymes (the transpeptidase from the bacteria the bacteria StreptomycesStreptomyces R61) that R61) that produce and then cross-link the strands produce and then cross-link the strands of this polymer together. This causes the of this polymer together. This causes the cell wall to lose strength and the bacteria cell wall to lose strength and the bacteria to burst. to burst.

Drug designDrug design is facilitated when an is facilitated when an enzyme that is essential to the enzyme that is essential to the pathogen's survival is absent or very pathogen's survival is absent or very different in humans. different in humans.

Humans do not make peptidoglycan, Humans do not make peptidoglycan, therefore inhibitors of this process are therefore inhibitors of this process are selectively toxic to bacteria. selectively toxic to bacteria.

Selective toxicity is also produced in Selective toxicity is also produced in antibiotics by exploiting differences in antibiotics by exploiting differences in the structure of the the structure of the ribosomesribosomes in in bacteria, or how they make bacteria, or how they make fatty acidsfatty acids

AcCHE, an enzyme found in animals from insects AcCHE, an enzyme found in animals from insects to humans. It is essential to nerve cell function to humans. It is essential to nerve cell function through its mechanism of breaking down the through its mechanism of breaking down the neurotransmitter neurotransmitter acetylcholineacetylcholine into its into its constituents, constituents, acetateacetate and and cholinecholine..

This is somewhat unique among This is somewhat unique among neurotransmitters as most, including neurotransmitters as most, including serotoninserotonin, , dopaminedopamine, and , and norepinephrinenorepinephrine, are absorbed , are absorbed from the from the synaptic cleftsynaptic cleft rather than cleaved. rather than cleaved.

Reversible competitive inhibitorsReversible competitive inhibitors, such as , such as

edrophoniumedrophonium, physostigmine, and neostigmine, , physostigmine, and neostigmine, are used in the treatment of myasthenia gravis are used in the treatment of myasthenia gravis and in anaesthesia. The carbamate pesticides are and in anaesthesia. The carbamate pesticides are also examples of reversible AChE inhibitors. also examples of reversible AChE inhibitors.

The organophosphate insecticides such as The organophosphate insecticides such as malathion, parathion, and chlorpyrifos malathion, parathion, and chlorpyrifos irreversibly inhibitirreversibly inhibit acetylcholinesterase. acetylcholinesterase.

ENZYME DEFICIENCIESENZYME DEFICIENCIES

MUCOPOLYSACCHARIDOSISMUCOPOLYSACCHARIDOSISMPS Type Eponym Deficient MPS Type Eponym Deficient

EnzymeEnzymeI H Hurler a-iduronidaseI H Hurler a-iduronidase

I H/S Hurler-Scheie a-iduronidaseI H/S Hurler-Scheie a-iduronidaseI S Scheie a-iduronidaseI S Scheie a-iduronidaseII Hunter Iduronidase sulfataseII Hunter Iduronidase sulfataseIII‡ Sanfilippo A Heparan sulfataseIII‡ Sanfilippo A Heparan sulfatase

Sanfilippo B Sanfilippo B NN-acetylglucosaminidase -acetylglucosaminidase Sanfilippo C Acetyl CoA glucosamine Sanfilippo C Acetyl CoA glucosamine

acetyltransferaseacetyltransferase Sanfilippo D Sanfilippo D NN-acetylglucosamine-6-sulfatase-acetylglucosamine-6-sulfataseIV Morquio A Galactosamine-6-sulfataseIV Morquio A Galactosamine-6-sulfatase Morquio BMorquio B b-galactosidase b-galactosidaseV NonexistentV NonexistentVI Maroteaux-Lamy VI Maroteaux-Lamy NN-acetylhexosamine-4-sulfatase-acetylhexosamine-4-sulfataseVII SlyVII Sly b-glucuronidase b-glucuronidaseIX Hyaluronidase HyaluronidaseIX Hyaluronidase Hyaluronidase

DeficiencyDeficiency

GLYCOGEN STORAGE GLYCOGEN STORAGE DISEASESDISEASES

TypeType - Enzyme Deficient- Enzyme Deficient -Clinical Features-Clinical Features

I I ((von Gierkevon Gierke)) - Liver and kidney - Liver and kidney Glucose-6-Glucose-6-phosphatasephosphatase Hepatomegaly, lactic acidosis, Hepatomegaly, lactic acidosis,

hyperlipidemia, severe fasting hypoglycemiahyperlipidemia, severe fasting hypoglycemia

II (Pompe)-II (Pompe)- All tissues’ All tissues’ alpha 1,4-glucosidasealpha 1,4-glucosidase

Cardiomegaly, muscle weakness, death in infancy and Cardiomegaly, muscle weakness, death in infancy and adultsadults

III (Cori-Forbes)-III (Cori-Forbes)- All tissues’ All tissues’ debrancher enzymedebrancher enzymeHepatomgaly, muscle weakness, fasting hypoglycermiaHepatomgaly, muscle weakness, fasting hypoglycermia

IV (Andersen)IV (Andersen)- All tissues’ - All tissues’ brancher 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

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

hypoglycemiahypoglycemia

VII (Tarui)VII (Tarui) Muscle and liver Muscle and liver phosphofructokinasephosphofructokinase

Pain and Pain and stiffness on stiffness on

exertionexertion

VIIIVIII Brain and liver Brain and liver adenylate kinaseadenylate kinase

Spasticity, Spasticity, decerebration, decerebration, high urinary high urinary

catecholamines, catecholamines, death in infancydeath in infancy

IXIX Liver phosphorylase Liver phosphorylase kinasekinase

Hepatomegaly, Hepatomegaly, occasional occasional

fasting fasting hypoglycemiahypoglycemia

XX Liver and muscle Liver and muscle cAMP-dependent cAMP-dependent

kinasekinase

Hepatomegaly Hepatomegaly onlyonly

LIPIDOSESLIPIDOSES

a group of inherited disorders a group of inherited disorders characterized by the characterized by the

accumulation of lipids in accumulation of lipids in tissuestissues especially the brainespecially the brain

due todue to

deficiency in a particular deficiency in a particular sphingolipid catabolic sphingolipid catabolic

enzymeenzyme

Niemann-Pick diseaseNiemann-Pick disease Deficiency in Deficiency in sphingomyelinasesphingomyelinase

and and accumulation of accumulation of sphingomyelinsphingomyelin

Gaucher’s diseaseGaucher’s disease Deficiency in Deficiency in -D-glucosidase-D-glucosidase and and

accumulation of accumulation of glucocerebrosideglucocerebroside

Krabbe’s diseaseKrabbe’s diseaseDeficiency in Deficiency in -D-galactosidase-D-galactosidase

and and accumulation ofaccumulation of galactocerebsidesgalactocerebsides

Fabry’s diseaseFabry’s disease Deficiency in Deficiency in -D-galactosidase-D-galactosidase

and and accumulation of ceramide accumulation of ceramide trihexosidetrihexoside

Tay-Sach’s diseaseTay-Sach’s disease Deficiency in Deficiency in -D-hexaminidase A-D-hexaminidase A

and and accumulation of ganglioside accumulation of ganglioside GMGM22

Metachromatic LeukodystrophyMetachromatic Leukodystrophy Deficiency in Deficiency in sulfatide sulfatasesulfatide sulfatase

and and accumulation ofaccumulation of --sulfogalactocerebrosidesulfogalactocerebroside