On Enzyme Nomenclature

and Classification 4

4.1 What Is in the Name?

The word enzyme (ενζυμη meaning in yeast in Greek), first used by Kuhne in 1877,
is now well accepted to describe a biological catalyst. Majority of enzyme names
today carry the suffix “-ase” as recommended for all enzyme names by Duclaux in
1898. Proteolytic enzymes are a significant exception to this generally accepted
norm. Some of them have retained the older tradition of usually ending with “-in,”
for example, trypsin, chymotrypsin, papain, and subtilisin.
All enzymes are proteins but not all proteins are enzymes. Catalysis by RNA
molecules (the so-called ribozyme) has expanded the realm of biological catalysts to
beyond proteins. While few RNA catalysts have been recognized, the vast majority
of enzymes that we come across in biology are proteins. Evolution has selected
L-isomers of 20 amino acids to build proteins. This has put limits on the number and
nature of available reactive chemical groups/functions that could be recruited for
catalysis. Proteins are rich in nucleophilic groups, but electrophiles are poorly
represented. In some instances, the protein component alone is inadequate to cata-
lyze a given reaction. Nature therefore recruited many nonprotein components called
cofactors to generate a functional catalyst. In such enzymes, the inactive protein
component without the cofactor is termed the apoenzyme and the active enzyme,
including the cofactor, the holoenzyme. The cofactors may be either metal ions (e.g.,
Mn[II] in arginase, Ni[II] in urease, and Ca[II] in DNase I) or coenzymes (organic
molecules like pyridine nucleotides NAD+ and NADP+, flavin adenine dinucleotide
(FAD), pyridoxal phosphate (PLP), thiamine pyrophosphate (TPP), biotin,
cobamide, and heme). Binding of a cofactor to its cognate apoenzyme could exhibit
a range of binding strength. A very tightly bound cofactor – which is difficult to
remove without damaging the enzyme – is also known as a prosthetic group. Quite
often prosthetic groups are covalently bound to the apoenzyme – lipoamide of
transacylase is an example.

# Springer Nature Singapore Pte Ltd. 2018 33

N. S. Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms,
https://doi.org/10.1007/978-981-13-0785-0_4
34 4 On Enzyme Nomenclature and Classification

Holoenzyme ¼ Apoenzyme þ Co factor

Non-covalent interactions between a protein (such as an apoenzyme) and a

cofactor may be weak or strong. This is amply obvious from the list of enzymes
that require divalent metal ions for activity. For instance, enzymes with a tightly
bound metal ion are termed metalloenzymes (such as urease), while those with a
weakly bound metal ion are grouped as metal-activated enzymes (such as Fe
[II]-catechol dioxygenase). Enzymes with metal ion dissociation constants (KD) of
the order of 108 M or higher are generally grouped as metal-activated enzymes,
while those with KD values lower than 108 M are considered metalloenzymes. This
boundary is artificial, and obviously there is a continuum of binding strengths
observed in nature. It may be noted in passing that even non-covalent interactions
can be very strong – essentially irreversible in few cases like avidin–biotin complex
(KD ¼ 1015 M; t1/2 of 2.5 years) or for that matter the two strands of a double-
stranded DNA!

4.2 Enzyme Diversity and Need for Systematics

The middle of the twentieth century saw an exponential increase in research on

enzymes. Sooner than later the number of new enzymes reported crossed manage-
able limits for an individual. As a consequence, in some cases, the same (or similar)
enzyme activities were given different names. Some names like catalase give very
little indication of the nature of the reaction they catalyze. Systematic classification,
cataloging, and nomenclature of enzymes therefore became a necessity. This was
easier recognized than done. Enzymes could be grouped according to any of the
following considerations:

(a) Occurrence and/or source of the enzyme: Laccase obtained from Japanese
lacquer tree, papain from papaya, and horseradish peroxidase are examples
of three such plant enzymes. Similarly a number of digestive enzymes are
isolated from pancreatic juice such as trypsin, chymotrypsin, carboxypepti-
dase, lipase, etc. the common source of lysozyme is from hen egg white
(HEW).
(b) Nature of the substrate on which the enzyme acts: They could be classified
into enzymes hydrolyzing (or acting on) proteins, carbohydrates, lipids, etc.
(c) Based on cofactor requirement: Typically many enzymes are simply pro-
teinaceous in nature. However those depending on a cofactor could be listed
into separate groups like thiamine pyrophosphate (TPP) enzymes, pyridoxal
phosphate (PLP) enzymes, metalloenzymes, etc.
(d) Common functional context: One could, in principle, group enzymes
belonging to discrete pathways like glycolytic enzymes, enzymes of histi-
dine biosynthesis, etc. they may also be grouped as soluble, membrane-
bound, or belonging to organelles such as mitochondria, etc.
4.3 Enzyme Commission: Recommendations 35

(e) Nature of the overall reaction catalyzed: An enzyme can be assigned to a

group by considering the type of the reaction it catalyzes. For instance, they
may catalyze oxidation, hydrolysis, etc.
(f) The mechanism of reaction: The intimate mechanism of the reaction at the
enzyme active site and the nature of intermediate complexes with the
enzyme may be considered. For example, proteases may be classified
depending upon whether an enzyme-bound acyl-enzyme intermediate is
formed or not.

It should be obvious from the above list that a systematic, meaningful classifica-
tion and cataloging of all the enzymes has not been easy. The problem is
compounded by the enormous diversity of enzyme structures and activities. A
typical RNA hydrolysis is achieved through a protein (RNase A), a protein-RNA
complex (RNase P) or RNA alone (ribozyme). The peptide bond hydrolysis is
possible with enzymes that are efficient in acidic pH or alkaline pH, require a
divalent metal ion, contain a serine -OH or cysteine -SH, etc. Enzymatic decarbox-
ylation of histidine may recruit pyridoxal phosphate or in a more primitive form may
simply use a bound pyruvate. Pyridoxal phosphate bound to glycogen phosphorylase
serves more of a structural role rather than function as a cofactor. Alkyl-
dihydroxyacetonephosphate synthase (an enzyme involved in the biosynthesis of
ether phospholipids) uses FAD for a non-redox reaction. Clearly, no single criterion
listed above would be satisfactory. To address these issues, an international com-
mission was set up in 1955 which presented its first report in 1961 (Table 2.1).

4.3 Enzyme Commission: Recommendations

Considering the diversity of enzyme sources, reactions, and mechanisms, it became

apparent that a formal system of nomenclature and classification was required.
“Enzyme Commission” was appointed by the International Union of Biochemistry
to address this issue. Its first report, published in 1964, forms the basis of present
system of classification. This system of classification is being updated periodically
with updates made in 1972, 1978, 1984, and 1992. There are also many electronic
supplements such as Supplement 14 of 2008 (Enzyme Nomenclature 1992). The
most recent information and guidelines on enzyme nomenclature may be found at
the official web site of the International Union of Biochemistry and Molecular
Biology (IUBMB) – http://www.chem.qmul.ac.uk/iubmb/enzyme/. The EC classifi-
cation is universally accepted with a unique name and EC number for each enzyme.
By this means names for every enzyme (listed with trivial name) could be
rationalized and also given an EC catalog number (McDonald et al. 2009).
The nature of the overall reaction catalyzed by the enzyme – expressed by the
formal equation – forms the basis of EC classification. Clearly the intimate mecha-
nism of the reaction and the formation of intermediate complexes with the enzyme, if
any, are not considered. The Enzyme Commission defined six general categories of
reactions, thereby assigning an enzyme to one of the six classes. A number of unique
36 4 On Enzyme Nomenclature and Classification

Fig. 4.1 Distribution of Ligases, 151

enzymes into six different
classes according to EC Isomerases,
classification. Data from 180 Oxido-
BRENDA database (January reductases,
2009 release) is shown Lyases, 462 1295
schematically

Hydrolases,
1476
Transferases,
1302

enzymes, represented in each of these six classes (BRENDA database release

January 2009), are shown in Fig. 4.1. Expectedly, enzymes for the redox and
hydrolytic reactions are the most represented group. These six classes are
oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Each of
these six classes is further divided into a number of subclasses and sub-subclasses,
according to the nature of the reaction catalyzed. In this classification each enzyme is
given a code number consisting of a four-number system. On this system the first
number indicates the main class and the second and third show the subclass and
sub-subclass, respectively, thus defining the type of reaction. The fourth number is
the actual number of that enzyme within its sub-subclass. For example, alcohol
dehydrogenase is given the code “EC 1.1.1.1.” The first number indicates that it
belongs to oxidoreductase class (EC 1.x.x.x). Within this class, enzymes acting on
CH-OH group of donors bear the same subclass number (EC 1.1.x.x). Within this
subclass, enzymes that use NAD or NADP as electron acceptor are given the number
EC 1.1.1.x. Since alcohol dehydrogenase is the first enzyme in this category, it gets
its fourth number (EC 1.1.1.1). Similarly, all carboxylesterases have the same first
three digits in their EC code (EC 3.1.1.x). The fourth digit however distinguishes
them by the actual carboxylic ester they hydrolyze.
Systematic name is assigned to each enzyme by the Commission, in addition to an
accepted trivial name. This name includes the name or names of substrates followed
by a reaction name that ends in “-ase.” Because such systematic names can at times
be too long and unwieldy, the Commission has also made recommendations for the
use of trivial names. However, for the group (EC 3.3.3.x) of common proteases like
pepsin, trypsin, papain, etc., it has not yet been possible to find acceptable systematic
names. Enzyme Commission nomenclatures for enzymes representative of each
class and those enzymes commonly referred to in this book are given in Table 4.1.
The universally accepted EC classification and enzyme codes are finding place
(and direct utility) in a number of databases describing enzymes, genes, genomes,
and metabolic pathways. Some of these databases are listed in Table 4.2.
Table 4.1 Enzyme commission nomenclatures for representative/common enzymes
EC No. Systematic name Trivial name Reaction
1. Oxidoreductases: Loss or gain of electrons by substrates
1.1.1.1 Alcohol:NAD+ oxidoreductase Alcohol dehydrogenase Alcohol+NAD+⇄ Aldehyde+NADH+H+
+
1.1.1.27 L-lactate:NAD oxidoreductase Lactate dehydrogenase L-lactate+NAD+⇄ Pyruvate+NADH+H+
1.2.1.2 Formate:NAD+ oxidoreductase Formate dehydrogenase Formate+NAD+⇄ CO2 + NADH+H+
1.4.1.4 L-Glutamate:NADP+ oxidoreductase NADP-glutamate L-Glutamate+H2O + NADP+⇄ 2-Oxoglutarate+NH3 + NADPH
(deaminating) dehydrogenase +H+
1.11.1.6 Hydrogen peroxide:Hydrogen peroxide Catalase H2O2+ H2O2⇄ O2 + 2H2O
oxidoreductase
2. Transferases: Transfer of a reactive group from a donor substrate to an acceptor substrate
2.1.2.1 L-Serine:Tetrahydrofolate 5,10- Serine L-Serine+Tetrahydrofolate⇄ Glycine+5,10-
hydroxymethyl-transferase hydroxymethyltransferase Methylenetetrahydrofolate
2.1.3.1 Methylmalonyl-CoA: Pyruvate Methylmalonyl-CoA Methylmalonyl-CoA + Pyruvate⇄ Propionyl CoA + Oxaloacetate
carboxytransferase carboxytransferase
4.3 Enzyme Commission: Recommendations

2.4.1.8 Maltose:Orthophosphate Maltose phosphorylase Maltose+Orthophosphate⇄ β-D-Glucose 1-phosphate+D-Glucose

glucosyltransferase
2.6.1.1 L-Aspartate:2-Oxoglutarate Aspartate aminotransferase L-Aspartate+2-Oxoglutarate⇄ Oxloacetate+L-Glutamate
aminotransferase
2.7. 1.1 ATP:D-Hexose 6-phosphotransferase Hexokinase ATP + D-Hexose⇄ ADP + D-Hexose 6-phosphate
2.7.7.16 Ribonucleate pyrimidine-nucleotido- Ribonuclease Transfers 30 -phosphate of a pyrimidine nucleotide residue of
20 -transferase (cyclizing) polynucleotide from 50 position of the adjoining nucleotide to the
20 position of the pyrimidine nucleotide itself, forms a cyclic
nucleotide
3. Hydrolases: Introduction of elements of water into a substrate
3.1.1.1 Carboxylic ester hydrolase Carboxylesterase Carboxylic ester+H2O⇄ Alcohol+Carboxylate
3.1.3.1 Orthophosphoric monoester Alkaline phosphatase Orthophosphoric monoester H2O⇄ Alcohol+Orthophosphate
phosphohydrolase
3.2.1.1 α-1,4-Glucan 4-glucanohydrolase α-Amylase Hydrolyzes α-1,4-glucan links in starch
3.2.1.17 Mucopeptide N-acetylmuramylhydrolase Lysozyme Hydrolyzes β-1,4-glucan links in peptidoglycan
37

3.4.4.4 Not possible yet Trypsin Hydrolyzes peptides, amides and esters of aromatic L-amino acids
(continued)
Table 4.1 (continued)
38

EC No. Systematic name Trivial name Reaction

3.5.3.1 L-Arginine amidinohydrolase Arginase L-Arginine+H2O⇄ L-Ornithine+Urea
3.7.1.1 Oxaloacetate acetylhydrolase Oxaloacetase Oxaloacetate+H2O⇄ Oxalate+Acetate
4. Lyases: Elimination of a group with double bond formation or addition of group to double bond
4.1.1.1 Pyruvate carboxy-lyase Pyruvate decarboxylase Pyruvate⇄ Acetaldehyde+CO2
4.2.1.1 Carbonate hydro-lyase Carbonic anhydrase H2CO3⇄ CO2 + H2O
4.3.1.1 L-Aspartate ammonia-lyase Aspartate ammonia-lyase L-Aspartate+⇄ Fumarate+NH3
4.3.2.1 L-Argininosuccinate arginine-lyase Argininosuccinate lyase L-Argininosuccinate⇄ Fumarate+L-arginine
4.99.1.1 Protoheme ferro-lyase Ferrochelatase Protoporphyrin+Fe2+⇄ Protoheme+2H+
5. Isomerases: Intramolecular rearrangements
5.1.1.4 Proline racemase Proline racemase L-Proline⇄ D-Proline
5.1.3.2 UDP-glucose 4-epimerase UDP-glucose epimerase UDP-glucose⇄ UDP-galactose
5.3.1.1 D-Glyceraldehyde-3-phosphate ketol- Triosephosphate isomerase D-Glyceraldehyde 3-phosphate⇄ Dihydroxyacetone phosphate
isomerase
5.3.1.5 D-xylose ketol-isomerase Xylose isomerase D-Xylose⇄ D-Xylulose
5.4.99.2 Methylmalonyl-CoA CoA-carbonylmutase Methylmalonyl-CoA Methylmalonyl-CoA⇄ Succinyl-CoA
mutase
6. Ligases (synthetases): Joining of molecules by covalent bond formation
6.1.1.1 L-Tyrosine:tRNA ligase (AMP) Tyrosyl-tRNA synthetase ATP + L-Tyrosine+tRNA⇄ AMP + Pyrophosphate+L-Tyrosyl-
tRNA
6.3.1.1 L-Aspartate:Ammonia ligase (AMP) Asparagine synthetase ATP + L-Aspartate+NH3⇄ AMP + Pyrophosphate+L-Asparagine
6.3.1.2 L-Glutamate:Ammonia ligase (ADP) Glutamine synthetase ATP + L-Glutamate+NH3⇄ ADP + Orthophosphate+L-Glutamine
6.3.4.5 L-Citrulline:L-Aspartate ligase (AMP) Argininosuccinate ATP + L-Citrulline+L-Aspartate⇄ AMP+ Pyrophosphate+L-
synthetase Argininosuccinate
6.4.1.2 Acetyl-CoA: Carbon dioxide ligase (ADP) Acetyl-CoA carboxylase ATP + Pyruvate+CO2 + H2O⇄ ADP+ Orthophosphate+Malonyl-
CoA
Enzyme examples selected in this table are those often referred to in this book
4 On Enzyme Nomenclature and Classification
4.4 Some Concerns 39

Table 4.2 Databases with enzyme EC numbers

Database Website
BRENDA (The Comprehensive Enzyme Information http://www.brenda-enzymes.info/
System)
ExPASy (Enzyme nomenclature Database) http://www.expasy.ch/enzyme/
KEGG (Kyoto Encyclopedia of Genes and Genomes) http://www.genome.ad.jp/kegg/
kegg2.html
MetaCyc (Pathway/Genome Databases) http://metacyc.org/ and http://biocyc.
org/
SYSTERS (Protein Family Database) http://systers.molgen.mpg.de/
InterPro (database of protein families, domains, and http://www.ebi.ac.uk/interpro/
functional sites)
Protein Mutant Database http://pmd.ddbj.nig.ac.jp/
BioCarta (Pathways of Life) http://www.biocarta.com/
ExplorEnz (The Enzyme Database) https://www.enzyme-database.org/
IUBMB (Enzyme Nomenclature) http://www.chem.qmul.ac.uk/iubmb/
enzyme/

4.4 Some Concerns

The EC system of classification and nomenclature was arrived from a broad consen-
sus with clear emphasis on the total reaction in question. The systematic names in a
given class may be based on a written reaction, even if only the reverse reaction is
experimentally demonstrated. This has created some situations that are less than
perfect. While all enzyme-catalyzed reactions are reversible in principle (at least
micro-reversibility at the active site!), the reaction-based classification for the for-
ward direction would not be the same as that for the reverse direction. This was
recognized very early by JBS Haldane, and according to him, calculation from
thermodynamic data shows that catalase may act in the direction of H2O2 synthesis
only under an O2 pressure of many billions of atmosphere! It would, therefore, be
perverse, if logical, to describe it as water oxidase or for that matter the peroxidase as
water dehydrogenase. To address such issues, the Commission has recommended
that the more important direction of the overall reaction, from a biochemical view
point, be used. It may be noted from Table 4.1 that reaction involving interconver-
sion of NADH and NAD+ is all written in the direction where NAD+ is reduced by
the other substrate. Also, when an overall reaction involves two types of reactions,
then the second function is indicated in brackets. For instance, an oxidoreductase
(decarboxylating) means the enzyme catalyzes an oxidation-reduction reaction in
which one of the substrates is being decarboxylated.
Apart from issues related to the direction of overall reaction considered, a major
concern is that the reaction mechanism is given less importance or completely
ignored! Functionally distinct reactions are catalyzed by the following four enzymes:
40 4 On Enzyme Nomenclature and Classification

Fumarate + H2O ⇄ Malate (Fumarate hydratase; EC 4.2.1.2)

Fumarate + NH3 ⇄ Aspartate (Aspartate ammonia-lyase; EC 4.3.1.1)
Fumarate + Arginine ⇄ Argininosuccinate (Argininosuccinate lyase; EC 4.3.2.1)
Fumarate + AMP ⇄ Adenylosuccinate (Adenylosuccinate lyase; EC 4.3.2.2)

All four enzymes are part of an evolutionary super-family of proteins; mechanis-

tically they add elements of H-X to fumarate, using Michael reaction. This is a
typical case where EC classification matches both in terms of overall reaction and the
reaction mechanism. While UDP-glucose epimerase – based on the overall reaction
– rightfully belongs to “isomerases” (Class 5), its intimate reaction mechanism tells a
different story. The enzyme has tightly bound NAD+, and the reaction at C4 of the
hexose involves redox chemistry. The reaction mechanism of glucosamine
6-phosphate isomerase is similar to that of ketose-aldose isomerase:

D-Glucosamine 6-phosphate þ H2 O⇄D-Fructose 6-phosphate þ NH3

It was therefore formerly grouped under Class 5 (with the code EC 5.3.1.10) as an
isomerase. Subsequently this reaction was recognized as a C-N bond hydrolysis and
reassigned as glucosamine 6-phosphate deaminase (EC 3.5.99.6).

Multiple Enzyme Forms and Isozymes At times we find that the same enzyme
activity in an organism (or different organisms) is displayed by different protein
forms. Enzymes that catalyze the same overall reaction but follow different mecha-
nistic paths are not uncommon. Some examples of different protein forms are listed
below (Table 4.3).

When these multiple molecular forms are coded by different but related genes
(having different primary structure – amino acid sequence!), they are termed isozymes.
Different isozyme forms of an enzyme are easily distinguished through their charac-
teristic electrophoretic mobilities. The muscle and the heart forms of lactate dehydro-
genase are the best studied examples of isozymes. All isozymes are examples of
multiple forms of an enzyme, but all multiple forms need not be isozymes.
Multiple molecular forms of the same enzyme may also arise due to other reasons.
They may occur as (a) interconvertible forms through covalent modifications (e.g.,
phosphorylation of glycogen phosphorylase and adenylylation of E. coli glutamine
synthetase), (b) proteolytic variants (chymotrypsinogen and chymotrypsin), (c) different
oligomeric states of the same monomer (e.g., bovine liver glutamate dehydrogenase and
avian liver acetyl CoA carboxylase), and (d) distinct conformational states of the same
enzyme protein (e.g., R and T states of aspartate carbamoyltransferase). Occurrence of
multiple enzyme forms is often associated with their role in metabolic regulation.
References 41

Table 4.3 Enzyme examples with different mechanistic forms

EC
Enzyme number Mechanistic difference
Ribonucleotide reductase EC An iron protein
1.17.4.1
EC Requires a cobamide coenzyme
1.17.4.2
Methionine synthase EC Contains cobalamin
2.1.1.13
EC Does not contain cobalamin
2.1.1.14
Proteases EC 3.4.4.x Serine, cysteine, carboxylate, or metal ion at active
site
Histidine decarboxylase EC Pyridoxal-phosphate (mammalian)
4.1.1.22 Pyruvoyl prosthetic group (bacterial)
Fructose bisphosphate EC Class I – Forms a protonated imine
aldolase 4.1.2.13 Class II – Zinc polarized (microbial)
Dehydroquinase EC Type I – Forms a protonated imine
4.2.1.10 Type II – Forms an enolate intermediate

All multiple enzyme forms share the same EC number and a formal name.
However these names need to be suitably prefixed or suffixed to indicate the
modification, organ source, or organelle source. Much effort goes into characterizing
any new enzyme. Therefore great care must be taken to define what it does and not
repeat the name or indicate another reaction. In the final outcome, one needs not get
bogged down by issues of semantics, for the excitement of enzymology beckons the
uninitiated and the specialist alike.

References
McDonald AG, Boyce S, Tipton KF (2009) ExplorEnz: the primary source of the IUBMB enzyme
list. Nucleic Acids Res 37:D593–D597
Enzyme Nomenclature 1992 [Academic Press, San Diego, California, ISBN 0-12-227164-5 (hard-
back), 0-12-227165-3 (paperback)] with Supplement 1 (1993), Supplement 2 (1994),
Supplement 3 (1995), Supplement 4 (1997) and Supplement 5 (in Eur J Biochem, 223:1–5
(1994); Eur J Biochem, 232:1–6 (1995); Eur J Biochem, 237:1–5 (1996); Eur J Biochem,
250:1–6 (1997) and Eur J Biochem, 264:610–650 (1999); respectively), IUBMB, Academic
Press, Orlando