Unit:1

The Enzyme Commission's system of classification

 The Enzyme Commission divided enzymes into six main classes, on the
basis of the total reaction catalysed.

 Each enzyme was assigned a code number, consisting offour elements,

separated by dots.

 The first digit shows to which of the main classesthe enzyme belongs, as
follows:

 First digit Enzyme classType of reaction catalysed(OTH LIL)

1 oxidoreductase Oxidation/Reduction reactions

2 Transferases Transfer of an atom or group between two molecules

(excluding reactions in other classes)

3 Hydrolases Hydrolysis reactions

4 Lyases Removal of a group from substrate

(not by hydrolysis)

5 Isomerases Isomerization reactions

6 Ligases The synthetic joining of two molecules, coupled with
the breakdown of the pyrophosphate bond in a
nucleoside triphosphate

 The naming and classification of enzymes:

 The second and third digits in the code further describe the kind of
reaction being
catalysed.

 There is no general rule, because the meanings of these digits are defmed
separately for each of the main classes.

 Example, in the case of the acyl group, which is transferred in reactions

catalysed by
E.C. 2.3 enzymes, it should be understood that the structure written -COR
Represents:

 Enzymes catalysing very similar but non-identical reactions, e.g. the

hydrolysis of
 different carboxylic acid esters, will have the same first three digits in
their code.

 The fourth digit distinguishes between them by defining the actual

substrate, e.g. the
 actual carboxylic acid ester being hydrolysed.
 However, it should be noted that isoenzymes, that is to say, different
enzymes
 catalysing identical reactions, will have the same four figure
classification.

 Thereare, for example, five different isoenzymes of lactate

dehydrogenase within the
human body and these will have an identical code.

 The classification, therefore,provides only the basis for a unique

identification of an enzyme.

 The particularisoenzyme and its source still have to be specified.

 It should also be noted that all reactions catalysed by enzymes are

reversible to some degree and the classification which would be given to
the enzyme for the catalysis of the forward reaction would not be the
same as that for the reverse reaction.

 The classification used is that of the most important direction from the
biochemical point of view, or according to some convention defined by
the Commission.

 For example, for oxidation/reduction involving the interconversion of

NADH and NAD+ (see section 11.5.2) the classification is usually based
on the direction where NAD+ is the electron acceptor rather than that
where NADH is the electron donor.

 The Enzyme Commission's recommendations on nomenclature:

 The Commission assigned to each enzyme a systematic name in addition
to its existing trivial name.

 This systematic name includes the name of the substrate or substrates in

full and a word ending in '-ase' indicating the nature of the process
catalysed.

 This word is either one of the six main classes of enzymes or a

subdivision of one of them.

 When a reaction involves two types of overall change, e.g. oxidation and
decarboxylation, the second function is indicated in brackets, e.g.
oxidoreductase (decarboxylating).

 Examples are given below.

 The systematic name and the Enzyme Commission (E.C.) classification

number unambiguously describe the reaction catalysed by an enzyme and
should always be included in a report of an investigation of an enzyme,
together with the source of enzyme, e.g. rat liver mitochondria.

 However, these names are likely to be long and unwieldy.

 Trivial names may,therefore, be used in a communication, once they have

been introduced and definedin terms of the systematic name and E.C.
number.
  Trivial names are also inevitablyused in everyday situations in the
laboratory.

 The Enzyme Commission maderecommendations as to which trivial

names were acceptable, altering those whichwere considered vague or
misleading.

 Thus, 'fumarase', mentioned above, wasconsidered unsatisfactory and was

replaced by 'fumaratehydratase'.

 The six main classes of enzymes

Main Class 1: Oxidoreductases

 These enzymes catalyse the transfer of H atoms, 0 atoms or electrons

from one substrate to another.

 The second digit in the code number of oxidoreductasesindicates the

donor of the reducing equivalents (hydrogen or electrons) involved inthe
reaction.

 For example:
Second digit Hydrogen or electron donor
1 alcohol (>CHOH)
2 aldehyde or ketone (>C=O)
3 --CH.CHprimary
4 amine (>CHNH2 or >CHNH3 "')
5 secondary amine (>CHNH-)
6 NADH or NADPH (only when some other
redox catalyst
is the acceptor

 The third digit refers to the hydrogen or electron acceptor, as follows:

Third digitHydrogen or electron acceptor

1 NAD+ or NADP+
2 Fe3+ (e.g. cytochrome)
3 02
99 An otherwise unclassified acceptor

 Trivial names of oxidoreductases include oxidases (transfer of H to 0 2)

and dehydrogenases (transfer ofH to an acceptor other than 02).

 These often indicate theidentity of the donor and/or acceptor. Here are
some examples:

 (S)-lactate: NAD+ oxidoreductase (E.C. 1.1.1.27), trivial name

lactatedehydrogenase, catalyses the reaction:

CH3.CH.co2 +NAD+↔CH3 C.CO2- + NADH + H+

I 11
OH O
(S)-lactate pyruvate

 Note that it is the alcohol group oflactate, rather than the carboxyl group,
which is involved in the reaction and this is indicated in the classification.
  Isocitrate: NAD+ oxidoreductase (decarboxylating) (E.C. 1.1.1.41),
trivial name isocitrate dehydrogenase.

 D-amino acid: oxygen oxidoreductase (deaminating) (E.C. 1.4.3.3),

trivial name Damino acid oxidase.

 Note that this enzyme is less specific than most and will act on any D-
amino acid.

Main Class 2: Trans/erases

 These catalyse reactions of the type:

AX+ B↔ BX + A

 but specifically exclude oxidoreductase and hydrolase reactions.

 In general, theEnzyme Commission recommends that the names of

transferases should end 'Xtransferase',whereXis the group transferred,
although a name ending 'trans-X-ase' is an acceptable alternative.

 The second digit in the classification describes the typeof group

transferred. For example:

Second digitGroup transferred

1 I -carbon group
2 aldehyde or ketone group (>C=O)
3 acyl group (--COR)
4 glycosyl (carbohydrate group)
7 phosphate group

 In general, the third digit further describes the group transferred. Thus:

E.C. 2.1.1 enzyme are methyltransferases (transfer --CH3), whereas

E.C. 2.1.2 enzymes are hydroxymethyltransferases (transfer --CH20H) and
E.C. 2.1.3 enzymes are carboxyl transferases (transfer --COOR)
 orcarbamoyltransferases (transfer --CONH2).
Similarly,

E.C. 2.4.1 enzymes are hexosyltransferases (transfer hexose units), and

E.C. 2.4.2 enzymes are pentosyltransferases (transfer pentose units).

 The exception to this general rule for transferases is where there is

transfer of phosphate groups: these cannot be described further, so there
is opportunity to indicate the acceptor.

E.C. 2.7.l enzymes are phosphotransferases with an alcohol group as acceptor,

E.C. 2.7.2 enzymes are phosphotransferases with a carboxyl group as acceptor,
E.C. 2.7.3 enzymes are phosphotransferases with a nitrogenous group as
acceptor.

 Phosphotransferases usually have a trivial name ending in '-kinase'. Some

examplesoftransferases are:

 ATP: D-hexose-6-phosphotransferase (E.C. 2.7.l.1) (trivial name:

hexokinase) whichcatalyses:
 C5H9O 5.CH20H + ATP~C5H90 5.CH20PO32-- + ADP
D-hexose D-hexose-6-phosphate

 This enzyme will transfer phosphate to a variety ofD-hexoses.

Main Class 3: Hydrolases

 These enzymes catalyse hydrolytic reactions of the form:

A-X + H20 ~ X-OH + HA

 They are classified according to the type of bond hydrolysed. For

example:

Second digitBondhydrolysed
1 ester
2 glycosidic (linking carbohydrate units)
4 peptide (-CONH-) (see chapter 2)
5 C-N bonds other than peptides

 The third digit further describes the type of bond hydrolysed. Thus:

E.C. 3.1.1 enzymes are carboxylic ester (-COO-) hydrolases,

E.C. 3.1.2 enzymes are thiol ester (-COS-) hydrolases,
E.C. 3.1.3 enzymes are phosphoric monoester ( -0 - Poi-) hydrolases,
E.C. 3.1.4 enzymes are phosphoric diester( -O-P02 -0-) hydrolases.

 For example, orthophosphoric monoester phosphohydrolase (E.C.

3.1.3.1) (alkaline phosphatase) catalyses:
 R-0-POj- + H20↔ R-OH + HO-PO32-
organic phosphate inorganic phosphate

 Alkaline phosphatases are relatively non-specific, and act on a variety of

substrates at alkaline pH.

 The trivial names of hydrolases are recommended to be the only ones to

consist simply of the name of the substrate plus '-ase'.

Main Class 4: Lyases

 These enzymes catalyse the non-hydrolytic removal of groups from

substrates, often
leaving double bonds.

 The second digit in the classification indicates the bond broken, for
example:

Second digitBond broken

1 C-C
2 C-0
3 C-N
4 C-S

 The third digit refers to the type of group removed. Thus, for the C-C
lyases:
Third digitGroup removed

1 carboxyl group (i.e. C02)

2 Aldehyde group (-CH=O)
3 ketoacid group ( -co.coz-)

 For example, L-histidinecarboxy-lyase (E.C. 4.1.1.22) (trivial name:

histidine
decarboxylase, catalyses:

C3N2H3.CH2CH.NH; ~ C3N2H3.CH2.CH2.NH; + C02 (1.10)

I
CO2
Histidine histamine

 (Note the importance of the hyphen and the extra 'y' in the systematic
name, becausecarboxy-lyase and carboxylase do not mean the same
thing: carboxylase simply refers to the involvement of C02 in a reaction
without being specific.)

 Also classified as lyases are enzymes catalysing reactions whose

biochemically important direction is the reverse of the above, i.e. addition
across double bonds.

 These may have the trivial name synthase or, if water is added across the
double bond, hydratase, as discussed earlier in the example of
fumaratehydratase(fumarase), the systematic name of this particular
enzyme being (S)-malate hydrolyase(E.C. 4.2.1.2).
Main Class 5: Isomerases

 Enzymes catalysing isomerization processes are classified according to

the type of
reaction involved. For example:

Second digit of reaction

1 Racemization or epimerization (inversion at an

asymmetric carbon atom)
2 cis – trans isomerization
3 intramolecularoxidoreductases
4 intramolecular transfer reaction

 The third digit describes the type of molecule undergoing isomerization.

Thus,
forracemases and epimerases:

Third digitSubstrate

1 amino acids
2 hydroxy acids
3 carbohydrates

 An example is alanine racemase (E.C. 5.1.1.1) which catalyses:

L-alanine↔ D-alanine

Main Class 6: Ligases

  These enzymes catalyse the synthesis of new bonds, coupled to the
breakdown of
ATP or other nucleotide triphosphates. The reactions are of the form:

X + Y + ATP ↔ X-Y + ADP + Pi

Or X + Y + ATP ↔ X-Y + AMP + (PP)i

 The second digit in the code indicates the type of bond synthesized. For
example:

Second digitBond synthesized

1 C-0
2 C-S
3 C-N
4 C-C

 The third digit further describes the bond being formed. Thus

E.C. 6.3.l enzymes are acid-ammonia ligases (amide, -CONH2 , synthases) and
E.C. 6.3.2 enzymes are acid-amino acid ligases (peptide, -CONH-, synthases).
Prior to 1984, such enzymes could also be known as synthetases.

 An example is L-glutamate: ammonia ligase (E.C. 6.3.1.2), trivial

name:glutamate-ammonia ligase, formerly glutamate synthetase, which
catalyses:
 O=C.CH2CH2.CH.C02 +ATP+ NH3 ~ O=C.CH2CH2 .CH.C02 +ADP
+Pi
II II
O NH2NH3
L-glutamate L-glutamine

PRATICAL ENZYMOLOGY
(A) Enzyme activity:
(B) Enzyme assay: