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Enzymes

Prof. Dr. Mohamed Fawzy Ramadan HassanienZagazig University, Egypt

Enzyme SpecificitySubstrate SpecificityReaction SpecificityStructureIsolation and PurificationMultiple Forms of EnzymesNomenclatureActivity Units

Enzyme Cofactors1-CosubstratesA-Nicotinamide Adenine Dinucleotide (NAD)B-Adenosine Triphosphate (ATP)2- Prosthetic GroupsA-FlavinsB- HeminC- Pyridoxal Phosphate3- Metal IonsMagnesium, Calcium and ZincB- Iron, Copper and Molybdenum

-Enzymes are proteins with powerful catalytic activity.

-They are synthesized by biological cells and in allorganisms, they are involved in chemical reactionsrelated to metabolism.

-Therefore, enzyme-catalyzed reactions also proceed inmany foods and thus enhance or deteriorate food quality.

-Relevant to this phenomenon are-the ripening of fruits and vegetables,-the aging of meat and dairy products,-the processing steps involved in the making of doughfrom wheat flours and-the production of alcoholic beverages by fermentationtechnology.

-Enzyme inactivation or changes in the distributionpatterns of enzymes in subcellular particles of tissue canoccur during storage or thermal treatment of food.

-Examples are the detection of pasteurization of milk,beer or honey, and differentiation between fresh anddeep frozen meat or fish.

-Enzyme properties are of interest to the food scientistsince enzymes are available in increasing numbers forenzymatic food analysis or for utilization in industrialfood processing.

General Remarks, Isolation and Nomenclature

Enzyme Specificity

-In addition to an enzyme’s ability to increase reactionrates, there is a unique enzyme property related to itshigh specificity for boththe compound to be converted (substrate specificity) andfor the type of reaction to be catalysed (reactionspecificity).

-The activities of allosteric enzymes are affected byspecific regulators or effectors. Thus, the activities ofsuch enzymes show an additional regulatory specificity.

Substrate Specificity

-The substrate specificity of enzymes shows the followingdifferences.

-The occurrence of a distinct functional group in the substrate isthe only prerequisite for few enzymes. This is exemplified bynon-specific lipases or peptidases which generally act on anester or peptide covalent bond.

-More restricted specificity is found in other enzymes, theactivities of which require that the substrate molecule contains adistinct structural feature in addition to reactive functional group.

-Examples are the proteinases trypsin and chymotrypsin whichcleave only ester or peptide bonds with the carbonyl groupderived from lysyl or arginyl (trypsin) or tyrosyl, phenylalanyl ortryptophanyl residues (chymotrypsin).

-Many enzymes activate only one singlesubstrate or preferentially catalyze theconversion of one substrate while othersubstrates are converted into products withlower reaction rate (examples in the Table).

-The reliable assessment of specificity ispossible only when the enzyme is availablein purified form.

-Another example is the specificity fordiastereoisomers, e.g. for cis-transgeometric isomers.

-Enzymes with high substrate specificity areof special interest for enzymatic foodanalysis. They can be used for the selectiveanalysis of individual food constituents,thus avoiding the time consumingseparation techniques.

Substrate specificity of a legume α-glucosidase

Reaction Specificity

-The substrate is specifically activated by the enzyme so that, among the severalthermodynamically permissible reactions, only one occurs.

-This is illustrated by the following example: L(+)-lactic acid is recognized as asubstrate by four enzymes although only lactate-2-monooxygenasedecarboxylates the acid oxidatively to acetic acid.

Examples of reaction specificity of some enzymes

Reaction Specificity (Cont.)

-Lactate dehydrogenase and lactate-malate transhydrogenase forma common reaction product, pyruvate, but by different reactionpathways.

-This may suggest that reaction specificity should be ascribed tothe different cosubstrates, such as NAD+ or oxalacetate.

-The enzyme’s reaction specificity as well as the substratespecificity are predetermined by the structure and chemicalproperties of the protein moiety of the enzyme.

-Of the four enzymes considered, only the lactate racemase reactswith either of the enantiomers of lactic acid, yielding a racemicmixture.

-Therefore, enzyme reaction specificity rather than substratespecificity is considered as a basis for enzyme classification andnomenclature.

Structure

-Enzymes are globular proteins with greatly differing particle sizes.

-As outlined previously, the protein structure is determined by its aminoacid sequences and by its conformation, both secondary and tertiary.

-Larger enzyme molecules often consist of two or more peptide chains(sub- units or protomers) arranged into a specified quaternary structure.

-It will be shown that the three dimensional shape of enzyme molecule isactually responsible for its specificity and its effective role as a catalyst.On the other hand, the protein nature of the enzyme restricts its activityto a relatively narrow pH range (for pH optima) and heat treatment leadsreadily to loss of activity by denaturation.

-Some enzymes are complexes consisting of a protein moiety boundfirmly to a non-protein component which is involved in catalysis, e.g. a“prosthetic” group.

-The activities of other enzymes require the presence of a cosubstratewhich is reversibly bound to the protein moiety.

Isolation and Purification

-Most of the enzyme properties are revealed only with purifiedenzymes.

-Prerequisites for the isolation of a pure enzyme are selectedprotein chemical separation methods carried out at 0-4 ◦C sinceenzymes are often not stable at higher temperatures.

Tissue Disintegration and Extraction-Disintegration and homogenization of biological tissue requiresspecial conditions and precautions.

-Procedures should be designed to rupture the majority of thecells in order to release their contents so that they becomeaccessible for extraction.

-The tissue is usually homogenized in the presence of anextraction buffer which often contains an ingredient to protect theenzymes from oxidation and from traces of heavy metal ions.

Isolation and Purification (Cont.)

-As a rule, large amounts of tissue have to be homogenized because the enzymecontent in proportion to the total protein isolated is low and is usually furtherreduced by the additional purification of the crude enzyme isolate (example inthe Table).

Isolation of α-glucosidase from beans (Phaseolus vidissimus)


Enzyme Purification-Removal of protein impurities is essentially the main approach inenzyme purification.

-As a first step, fractional precipitation, e.g. by ammonium sulfatesaturation, is often used or the extracted proteins are fractionated bymolecular weight , e.g., column gel chromatography.

-The fractions containing the desired enzyme activity are collected andpurified further, e.g., by ion-exchange chromatography.

-Additional options are also available, such as various forms ofpreparative electrophoresis, e.g. disc gel electrophoresis or isoelectricfocusing.

-The purification procedure can be shortened by using affinity columnchromatography. In this case, the column is packed with a stationaryphase to which is attached the substrate or specific inhibitor of theenzyme. The enzyme is then selectively and reversibly bound and, thus,in contrast to the other inert proteins, its elution is delayed.


Control of Purity-Previously, complete removal of protein impurities was confirmedby crystallization of the enzyme. This “proof” of purity was opento criticism.

-Today, electrophoretic methods of high separation efficiency orHPLC are used.

-The behavior of the enzyme during chromatographic separation isan additional proof of purity. A purified enzyme is characterized byan elution peak in which the positions of the protein absorbanceand enzyme activity coincide and the specific activity remainsunchanged during repeated elutions.

-During purification procedure, the enzyme activities are recorded.They show the extent of purification achieved after eachseparation step and show the enzyme yield.

Multiple Forms of Enzymes-Chromatographic or electrophoretic separations of enzymes can resultin separation of the enzyme into “isoenzymes”, i.e. forms of the enzymewhich catalyze the same reaction although they differ in their proteinstructure.

The occurrence of multiple enzyme forms can be result of the following:

a) Different compartments of the cell produce genetically independent enzymeswith the same substrate and reaction specificity, but differ in their primarystructure.An example is glutamate-oxalacetate transaminase found in mitochondria andalso in muscle tissue sarcoplasm. This is the indicator enzyme used todifferentiate fresh from frozen meat.

b) Protomers associate to form polymers of differing size.An example is the glutamate dehydrogenase found in tissue as an equilibriummixture of different molecular weights.

c) Different protomers combine in various amounts to form the enzyme.For example, lactate dehydrogenase is structured from larger number ofsubunits with the reaction specificity. It consists of five forms (A4 , A3 B, A2 B2 ,AB3 and B4 ), all derived from two protomers (A and B).

Nomenclature-The Nomenclature Commitee of the “International Union ofBiochemistry and Molecular Biology” (IUBMB) adopted rules in 1992 forthe systematic classification and designation of enzymes based onreaction specificity.

-All enzymes are classified into six major classes according to the natureof the chemical reaction catalyzed:

1.Oxidoreductases.2.Transferases.3. Hydrolases.4.Lyases (cleave C−C, C−O, C−N, and other groups by elimination, leaving

double bonds, or conversely adding groups to double bonds).

5.Isomerases (involved in the catalysis of isomerizations within onemolecule).

6.Ligases (involved in the biosynthesis of a compound with the simultaneous

hydrolysis of a pyrophosphate bond in ATP or a similar triphosphate).

-Each class is then subdivided into subclasses which denote thetype of reaction, e.g. by naming the electron donor of anoxidation-reduction reaction or by naming the functional groupcarried over by transferase or cleaved by hydrolase enzyme.

-Each subclass is further divided into sub-subclasses. Forexample, sub-subclasses of oxidoreductases are denoted bynaming the acceptor which accepts the electron from itsrespective donor.

-Each enzyme is classified by adopting this system.

-The enzyme ascorbic acid oxidase catalyzes the followingreaction:

-Its systematic name is L-ascorbate oxygen oxidoreductase, andits systematic number is E.C. 1.1.10.3.3.

-The systematic names are often quite long. Therefore, the trivialnames along with the systematic numbers are often convenientfor enzyme designation.

-Since enzymes of different biological origin often differ in theirproperties, the source and, when known, the subcellular fractionused for isolation are specified in addition to the name of theenzyme preparation;for example, “ascorbate oxidase from cucumber”. When known,the subcellular fraction of origin (cytoplasmic, mitochondrial) isalso specified.

-A number of enzymes of interest to food chemistry aredescribed in the following Table.

-The number of the section in which an enzyme is dealt with isgiven in the last column.

Systematic classification of enzymes of importance to food chemistry

Activity Units-The catalytic activity of enzymes is exhibited only under specific conditions, such as pH,ionic strength, buffer type, presence of cofactors and suitable temperature. Therefore,the rate of substrate conversion or product formation can be measured in a test systemdesigned to follow the enzyme activity.

-The International System of Units (SI) designation is mol.s−1 and its recommendeddesignation is the “katal” (kat* ).

Decimal units are formed in the usual way, e. g.:

-Concentration of enzymatic activity is given as μkat l−1 .

The following activity units are derived from this:

a) The specific catalytic activity, i.e. the activity of the enzyme preparation in relation tothe protein concentration.

b) The molar catalytic activity. This can be determined when the pure enzyme with aknown molecular weight is available.-It is expressed as “katal per mol of enzyme” (kat mol−1 ). When the enzyme has onlyone active site or center per molecule, the molar catalytic activity equals the “turnovernumber”, which is defined as the number of substrate molecules converted per unit timeby each active site of the enzyme molecule.

Enzyme Cofactors

-Numerous enzymes are not pure proteins and contain metal ionsand/or low molecular weight nonprotein organic molecules.

-These nonprotein constituents are denoted as cofactors which areessential for enzyme activity.

-According to the systematics,an apoenzyme is the inactiveprotein without a cofactor.

-Metal ions and coenzymesparticipating in enzymaticactivity belong to thecofactors which aresubdivided into prostheticgroups and cosubstrates.

-The prosthetic group isbound firmly to the enzyme. Itcan not be removed by, e. g.dialysis, and during enzymecatalysis it remains attachedto the enzyme molecule.

Systematics of cofactor-containingenzymes

-Often, two substrates are converted by such enzymes, onesubstrate followed by the other, returning the prosthetic group toits original state.

-On the other hand, during metabolism, the co-substrate reactswith at least two enzymes. It transfers the hydrogen or thefunctional group to another enzyme and, hence, is denoted as a“transport metabolite” or as an “intermediary substrate”.

-It is distinguished from a true substrate by being regenerated in asubsequent reaction. Therefore, the concentration of theintermediary substrates can be very low.

-In food analysis higher amounts of co-substrates are used withoutregeneration.

-Some cofactors are related to water-soluble vitamins.

1-CosubstratesA-Nicotinamide Adenine Dinucleotide (NAD)

-Nicotinamide adenine dinucleotide, abbreviated NAD+, is a coenzymefound in all living cells.

-The compound is a dinucleotide, since it consists of two nucleotidesjoined through their phosphate groups. One nucleotide contains anadenine base and the other nicotinamide.

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP);

R = H: NAD; R = PO3H2 : NADP

A-Nicotinamide Adenine Dinucleotide (NAD)

-In metabolism, NAD+ is involved in redox reactions, carryingelectrons from one reaction to another.

-The coenzyme is, therefore, found in two forms in cells:

NAD+ is an oxidizing agent– it accepts electrons from othermolecules and becomes reduced.

-This reaction forms NADH, which can then be used as a reducingagent to donate electrons.

The redox reaction of NAD

A-Nicotinamide Adenine Dinucleotide (NAD)

-The oxidized and reducedforms of the pyridine cosubstrateare readily distinguished byabsorbance readings at 340 nm.

-Therefore, whenever possible,enzymatic reactions which aredifficult to measure directlyare coupled with an NAD(P)-dependent indicator reaction forfood analysis.

Electron excitation spectra of NAD (1) and NADH (2)

1-CosubstratesB-Adenosine Triphosphate (ATP)

The nucleotide adenosine triphosphate(ATP) is an energy-rich compound.

-Various groups are cleaved and transferredto defined substrates during metabolism inthe presence of ATP.

-One possibility, the transfer oforthophosphates by kinases, is utilized inthe enzymatic analysis of food.

-Riboflavin, known as vitamin B2, isthe building block of flavinmononucleotide (FMN) and flavinadenine dinucleotide (FAD).

-Both act as prosthetic groups forelectron transfer reactions in a numberof enzymes.

-Due to the much wider redoxpotential of the flavin enzymes,riboflavin is involved in the transfer ofeither one or two electrons. This isdifferent from nicotinamides whichparticipate in double electron transferonly.

2- Prosthetic GroupsA-Flavins

Flavin adenine dinucleotide(FAD)

-An example for a flavin enzyme is glucoseoxidase, an enzyme often used in foodprocessing to trap residual oxygen.

-The enzyme isolated and purified fromAspergillus niger with two noncovalentlybound FAD molecules.

-In contrast to xanthine oxidase, thisenzyme has no heavy metal ion.

-During oxidation of a substrate, such asthe oxidation of glucose to gluconolactone,the flavoquinone is reduced by two singleelectron transfers.


-Like glucose oxidase, many flavin enzymes transfer the electronsto molecular oxygen, forming H2O2 and flavoquinone.

-The following intermediary products appear in this reaction:


2- Prosthetic GroupsB- Hemin

-Peroxidases from food of plant origin and several catalasescontain ferri-protoporphyrin (hemin) as their prosthetic group andas the chromophore responsible for the brown color of theenzymes.

-In catalytic reactions there is a change in the electron excitation spectra of theperoxidases (Fig.a) which is caused by a valence change of the iron ion (Fig. b).

-Intermediary compounds I (green) and II (red) are formed during this change byreaction with H2O2 and reducing agent AH.

-Some verdoperoxidases, which are green in color and found in many foods ofanimal origin, e.g. milk, contain an unidentified Fe-protoporphyrin as theirprosthetic group.

Peroxidase reaction with H2O2 and a hydrogen donor (AH). a Electron excitation spectra of peroxidase and intermediates I and II;

b mechanism of catalysis

2- Prosthetic GroupsB-Hemin

2- Prosthetic GroupsC- Pyridoxal Phosphate

Pyridoxal phosphate

and pyridoxamine

are designated as vitamin B6 and are essential ingredients of food


-Coupled to the enzyme as a prosthetic group through a lysyl residue,pyridoxal phosphate is involved in conversion reactions of amino acids.

-In the first step of catalysis, the amino group of the amino acidsubstrate displaces the 6-amino group of lysine from the aldiminelinkage.

-The positively charged pyridine ring then exerts an electron shifttowards the α-C-atom of the amino acid substrate; the shift beingsupported by the release of one substituent of the α-C-atom.


-This Figure shows how theionization of the protonattached to the α-C-atomleads to transamination of theamino acid with formationof an α-keto acid.

-The reaction may alsoproceed through adecarboxylation and yield anamine.Which of these two pathwayoptions will prevail is decidedby the structure of the proteinmoiety of the enzyme.

The role of pyridoxal phosphate in transamination and decarboxylation of

amino acids

3- Metal Ions

-Metal ions are essential cofactors and stabilizers of theconformation of many enzymes.

-They are especially effective as cofactors with enzymesconverting small molecules.

-They influence the substrate binding and participate incatalytic reactions or play the role of an electron carrier.

-Only the most important ions will be discussed.

3- Metal IonsA- Magnesium, Calcium and Zinc

-Mg2+ ions activate some enzymes which hydrolyze phosphoric acid esterbonds (e.g. phosphatases) or transfer phosphate residues from ATP to asuitable acceptor (e.g. kinases).

-In both cases, Mg2+ ions act as an electrophilic acid, polarize the P−O-linkageof the phosphate residue of the substrate or cosubstrate and, thus, facilitate anucleophilic attack (water with hydrolases).

-An example is the hexokinase enzyme which, in glycolysis, is involved incatalyzing the phosphorylation of glucose to glucose-6-phosphate with ATP ascosubstrate.

-The effect of a Mg2+ ion within the enzyme-substrate complex is obvious fromthe following formulation


-Ca2+ ions are weaker than Mg2+ ions. Therefore, the

replacement of Mg2+ by Ca2+ may result in an inhibition of thekinase enzymes.

-Enhancement of the activity of other enzymes by Ca2+ is basedon the ability of the ion to interact with the negatively chargedsites of amino acid residues and, thus, to bring aboutstabilization of the enzyme conformation (e.g. α-amylase).

-The activation of the enzyme may be also caused by theinvolvement of the Ca2+ ion in substrate binding (e.g. lipase).


-Zn2+ ion is a cofactor which is not involved in redox reactions underphysiological conditions. Mg2+, Zn2+ are similar in strength, hence,substituting the Zn2+ ion for the Mg2+ ion in some enzymes is possiblewithout loss of enzyme activity.

-Both metal ions can function as stabilizers of enzyme conformation andtheir direct participation in catalysis is readily revealed in the case ofalcohol dehydrogenase.

-This enzyme isolated from horse liver consists of two identicalpolypeptide chains, each with one active site. Two of the four Zn2+

ions in the enzyme readily dissociate. Although this dissociation has noeffect on the quaternary structure, the enzyme activity is lost.

-In catalysis they polarize the substrate’s C−O linkage and, thus, facilitatethe transfer of hydride ions from or to the cosubstrate.

3- Metal IonsB- Iron, Copper and Molybdenum

-The redox system of Fe3+/Fe2+ covers a wide range of potentialsdepending on the attached ligands. Therefore, the system isexceptionally suitable for bridging large potential differences in astepwise electron transport system.

-Such an example is encountered in the transfer of electrons bythe cytochromes as members of the respiratory chain or in thebiosynthesis of unsaturated fatty acids, and by some individualenzymes.

-Iron-containing enzymes are attributed either to the heme or tothe non-heme Fe-containing proteins (lipoxygenase and xanthineoxidase).


-Xanthine oxidase from milk reacts with many electron donors andacceptors. However, this enzyme is most active with substrates such asxanthine or hypoxanthine as electron donors and molecular oxygen asthe electron acceptor.

-The enzyme is assumed to have two active sites per molecule, witheach having 1 FAD moiety, 4 Fe-atoms and 1 Mo-atom.

-During the oxidation of xanthine to uric acid, oxygen is reduced by twoone-electron steps to H2O2 by an electron transfer system in which thefollowing valence changes occur


-Polyphenol oxidases and ascorbic acid oxidase, which occur infood, are known to have a Cu2+/Cu1+ redox system as a prostheticgroup.

-Polyphenol oxidases play an important role in the quality offood of plant origin because they cause the “enzymaticbrowning” for example in potatoes, apples and mushrooms.

-Tyrosinases, catecholases or phenolases are enzymes that reactwith oxygen and a large range of mono and diphenols.

-Polyphenol oxidase catalyzes two reactions:first the hydroxylation of a monophenol to o-diphenol(monophenol monooxygenase) followed by an oxidation to o-quinone (oxygen oxidoreductase).

B- Iron, Copper and Molybdenum

-At its active site, polyphenol oxidase contains two Cu1+ ions with two histidineresidues each in the ligand field.

-In an “ordered mechanism” the enzyme first binds oxygen and latermonophenol with participation of the intermediates (shown in the Figure).

-The Cu ions change their valency (Cu1+ →Cu2+).

-The newly formed complex ([] in the Figure) has a strongly polarizedO−O=bonding, resulting in a hydroxylation to o-diphenol. The cycle closes withthe oxidation of o-diphenol to o-quinone.

Mechanism of polyphenol oxidase activity

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