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INTRODUCTION TO ENZYMOLOGY
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INTRODUCTION TO ENZYMES
Enzymes
Gntienzymes
ëm Gntienzymes are those substances which when injected in the body produces
certain molecules, which act as inhibitors and inhibits the function of the
enzyme, related to that particular reaction
ëm Examples: Gntitrypsin, antirennin, antipepsin, etc.
strate
ëm dhe substrate is a substance upon which an enzyme acts and gets converted into
the corresponding product
ëm or example, maltose is the substrate over which the enzyme maltase acts to
from glucose
Maltase
Maltose Glucose
O aracteristics of Enzymes
1m Colloidal nature
ëm dhey are of great size
ëm dheir molecular weights usually range from 12000 to over a million Daltons
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ëm Hence, they are very large compared to their substrates or the functional
group they act upon
ëm dhe molecular weight of many enzymes are found to be approximately 6-
fold (6 is an integer multiple of 17500, which is found to be a unit in most
proteins
ëm n account of their large size, the enzyme molecules possess extremely low
rates of diffusion and forms colloidal systems in water
ëm Due to their giant size, the enzymes exhibit many colloidal properties such
as:
i m Diffusion rates are very slow
ii m May produce considerable high light scattering. dhey form turbidity in
solution known as dyndall effect
2m Catalytic nature
ëm G universal feature of all enzymatic reactions is the virtual absence of any
side product
ëm Gn enzyme is precisely adapted to catalyze a particular reaction. or
example, amylase catalyzes the breakdown of starch
ëm dhey act catalytically and accelerate the rate of chemical reactions,
occurring in the plant and animal tissues
ëm dhey normally do not participate in the reaction, or if they do so, at the end
of the reaction, they are recovered as such without undergoing any
qualitative or quantitative change
ëm dhis is why they are capable of catalyzing the transformation of a large
quantity of substrate
ëm dhus, the catalytic potency of enzymes is extremely great
3m durnover number
ëm dhe catalytic power of an enzyme is measured by the turnover number or the
molecular activity
ëm t is defined as the number of substrate molecules converted into the product
in a given unit of time by a single enzyme molecule, when the enzyme is
fully saturated with the substrate
ëm dhe turnover number of some enzymes are given as follows:
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ëm durnover number is represented as Kcat. dhe constant Kcat is a first order rate
constant and its unit is s61
max
KV
[E]d
ëm dhe value of the turnover number varies with different enzymes and it
depends upon the conditions in which the reaction is taking place
ëm dhe turnover number of 40,000,000 s61for carbonic anhydrase is one of the
largest known
ëm Carbonic anhydrase catalyzes the hydration of carbon dioxide to form
carbonic acid:
Carbonic anhydrase
CO2 + 2 O 2 CO3
ëm dhis catalyzed reaction is 4×107 times faster than the uncatalyzed one
ëm or most enzymes this value falls between 1±104 s61
4m Specificity of enzyme action
ëm Enzymes are highly specific in their action when compared with a chemical
catalytic reaction; i.e., a particular enzyme attacks only a particular substrate
ëm ccurrence of thousands of enzymes in the biological system is due to the
specific nature of enzymes
ëm Enzyme specificity is based on the mode of accepting the substrate and their
reaction
ëm dhey are
i m Stereochemical or optical specificity
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Gsp and so on. Each reaction of GG is catalyzed by its own separate
enzyme, which catalyzes only that reaction and none other
Gspartate
dransaminase
§
Decarboxylation
Citrate
Gcetylation
CH 3CH
xaloacetate
Decarboxylase
yruvate
Gcetylase
Reductase
Malate
Gln
Glutaminase
§
Gsp -KG
Gspartate aminotrasferase Glanine aminotransferase
Glu Gla -KG
Reductase
Malate
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(4 m artly due to its specificity and partly because they take place at
relatively lower temperatures, enzyme catalyzed reactions are much
more quantitative
iii mSubstrate specificity
(1 mdhe extent of substrate specificity varies from one enzyme to the other
(2 mt may be either absolute or relative
(3 mGbsolute specificity (one to one specificity is seen in some enzymes
which are capable of acting on only 1 substrate. or example, urease
acts only upon urea
(4 mRelative specificity can be further classified into group dependant or
bond dependent
(5 mGroup substrate specificity
(a mGbsolute group specificity or relative group specificity or broad
specificity
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(iii dhrombin splits peptide bonds in which the side chain on the
carboxyl site of the susceptible peptide bond must be Grg, while
the one on the amino group must be Gly
iv mGeometrical specificity
(1 mSome enzymes exhibit specificity towards the cis/trans forms
(geometric isomers
(2 mor example, fumarase catalyzes the interconversion of fumarate
(trans form and malate (cis form
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5m Gmphoteric nature
ëm Enzymes are amphoteric. dhey act as both acids and bases
ëm dhey migrate in an electric field and the direction of their migration depends
upon the charge possessed by them
ëm dhe net charge is influenced by their pH value
ëm Each enzyme has a fixed value of isoelectric point (p at which it will move
in an electric field
ëm soelectric field or isoelectric point is the pH value at which the number of
cations equals the number of anions
ëm dhus, at p, the net electric charge on an enzyme is always 0
ëm But, the total charge on an enzyme molecule (sum of the positive and
negative charges at this point is always maximum
ëm Hence, enzymes are dipolar ions (or internal salts (or zwitterions
ëm Gt p, they exist as 3 ( OO n
ëm Gt pH values m p, enzymes will have a net positive charge and will migrate
towards the cathode
ëm Similarly, at pH ´ p, enzymes will have a net negative charge and will
migrate towards the anode
Acidic behaviour
2
OO 2 OO +
asic behaviour
2
OO 3
OO + l
+ l
6m Solubility
ëm dhe solubility of enzymes is markedly influenced by the pH
ëm Solubility is lower at p and increases with increasing acidity or alkalinity
ëm n either cation or anion, repulsive forces between enzymes are high, since
all the molecules possess excess charge of the same site. dhus they will be
more soluble
ëm Salting-in effect
i m Globulins are slightly soluble in water.
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ëm Reversible denaturation
ëm rreversible denaturation
ëm Significance of denaturation
i m Denaturation property of a protein is harmful in clinical laboratories
ii m dhe protein-free substance of the blood such as glucose, picrate, and
drugs are analyzed by the precipitation of blood proteins by the addition
of certain acids
9m Reversibility of a Reaction
ëm Enzymes are capable of bringing about reversion in a chemical reaction
ëm dhe digestive enzymes catalyze the hydrolytic reactions which are reversible
ëm or example, lipase, which catalyzes the synthesis of fat from glycerol and
fatty acids, can also hydrolyze them into their component units
2 O
ipase
( 2 OO 15 31 3 +3 2
O O +3 15 31 OO
2 O
dripalmitin Water lycerol Palmitic acid
ëm dhe direction in which the reaction proceeds depends upon many factors like
(1 mpH of cell sap
(2 mresence of reacting substance
(3 mGccumulation of end products
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ëm dhe final equilibrium mixture is the same, whether the reaction starts with
the ester, or with its individual components
ëm Each enzyme has its own tertiary structure and specific conformation, which
is very essential for its catalytic activity
ëm Chemically, enzymes may be divided into 2 categories
ëm Simple protein enzymes
§m dhese contain simple proteins only. E.g., urease, amylase, papain, etc.
ëm Complex protein enzymes
§m dhese contain conjugated proteins, i.e., they have a protein part called
apoenzyme and a non-protein part called prosthetic group, associated
with the protein unit
§m dhe 2 parts together constitute the holoenzyme. E.g., catalase,
cytochrome V, etc.
ëm Holoenzyme
§m dhe functional unit or the active structure of an enzyme
(apoenzyme prosthetic group is called holoenzyme
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ëm Gpoenzyme
§m t is the protein part of the enzyme
§m t is the inactive form of the enzyme
§m t becomes functional only by associating itself with the prosthetic
group
ëm rosthetic group
§m G prosthetic group is that which is covalently bound to the apoenzyme
§m dhey do not dissociate from the protein part of the enzyme and
repeatedly participate in enzyme-catalyzed reactions
§m E.g., GD, MN, d, L, biotin, etc.
ëm Cofactor
§m dhey are mainly inorganic metal complexes which are tightly bound
to the enzyme
§m dhey are highly required for normal conformational structure and
function of the enzyme
§m dhey act as
donors
or acceptors
in oxidation
and reduction
reactions
2 2 2 2 2 2 2
§m E.g., Mg , Ca , Cu , Zn , K , e , Mn , Mo , etc.
ëm Coenzymes
§m dhey are also called co-substrate or second substrate
§m dhey are organic, metallo-organic, or inorganic substances which are
thermostable and dialyzable, highly required for the normal
functioning of the enzyme
§m dhey bind covalently or non-covalently to the apoenzyme
§m Reactions involving oxidoreductions, group transfers, isomerization,
and covalent bond formation require coenzyme
§m Coenzymes account for 1% of the entire enzyme molecule
§m E.g., NGD, GD, dH, CoG. MN, d, etc.
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Olassification of Ooenzymes
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nctions of Ooenzymes
ëm dhe activity
of many
enzymes
depend on the presence of many metal ions such
2 2 2 2
as Mg ,Ca , Cu , Zn , K , etc.
ëm Depending upon the interaction between the enzyme molecule and the metal
ion, the enzymes are classified as metal activated enzymes and metalloenzymes
ëm Metal activated enzymes
§m n certain enzymes, metals form a loose and easily dissociable complex
§m Such enzymes are called metal activated enzymes
§m dhe metal ion can be removed by dialysis or any other simple method
from the enzymes without causing denaturation (inactivation of
apoenzyme
§m or example, Gdase±Mg2 and Ca2 , enolase±Mg2 , etc.
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ëm Metalloenzymes
§m Some enzymes which are tightly bound to metal ions are called
metalloenzymes
§m dhese metals cannot be dissociated from the apoenzyme, even after
several extensive steps of purification
§m or example, Cu2 cytochrome oxidase,phenol oxidase; Zn
2
carbonic
2 2
anhydrase, alcohol dehydrogenase; Mg hexokinase; Ni urease, etc.
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M
yruvate kinase Gd
Creatine
ëm Substrate bridge complexes
§m dhe formation of ternary substrate bridge complexes of nucleoside
triphosphates with enzyme, metal and substrate appears to be attributed
to the displacement of water from the coordination sphere of the metal by
Gd
2
Gd 46 M(H 2 26
> >> > Gd 6 M(H 2 3 3H 2
6
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§m or binary E±M complexes, the rate limiting step is the departure of
water from the coordination sphere of the metal ion
§m or many peptidases, activation by metal ions is a slow process requiring
many hours
§m dhe slow reaction probably is due to the conformational rearrangement of
the binary E±M complex to an active conformation
E M( 2
apid
O 6 E M( 2 O 6 6 6 2 O
ëm Enzymes, which are synthesized in a particular cell and are transported to the
target site where they catalyze biochemical reactions, are called extracellular
enzymes
ëm dhey are also known as exoenzymes
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ëm dhey can be converted into the active form (zymase form by modifications in
their peptide bonds
ëm or example,
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ëm Enzymes which have only one polypeptide chain in their structre are known as
monomeric enzymes
ëm Examples: RNase, DNG polymerase , E, pyruvate carboxylase
ëm When many different enzyme-catalyzing reaction sites are lovated at different
sites of the same molecule, it is known as a multimeric or multienzyme
complex
ëm dhe complex becomes inactive when it is freactionated into smaller units, each
bearing individual enzyme activity
ëm dhese complexes pass intermediate products along from enzyme-to-enzyme by
transfer reactions
ëm Examples: atty acid synthetase, CS , DH, G synthase
Enzyme d rnover
ëm dhe requirement for the formation of a single intermediate complex can include
the reaction of the type:
+ > >> >1> >
2
P
3
+P
1
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K 2 [E 0 ] [S]
0 =
K m [S]
but E 0 [E] [S]
Km
[ES]
[E] [S]
K
0 cat [E] [S]
Km
soenzymes (sozymes)
ëm Enzymes which exist in tissues in two or more forms and have the same
catalytic activity, but are physically, chemically, immunologically, and
electrophoretically distinct are known as isoenzymes or isozymes
ëm dhey are present in the serum and tissues of mammals, amphibians, birds,
insects, plants and unicellular organisms
ëm Examples include isozymes of numerous dehyrogenases, several oxidases,
transaminases, phosphatases, transphosphorylases, proteolytic enzymes,
aldolases, etc.
O
ëm dhey catalyze the same reaction, but can be distinguished by physical methods
like electrophoresis or immunological techniques
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ëm dhe difference between some isozymes are due to differences in the quaternary
structure of the enzyme
ëm or example, lactate dehydrogenase (LDH exist in 5 isozymic forms
ëm dhe isozymic forms of LDH are tetramers, each made up from 2 tyes of units H
and M. dhe molecular weight of active LDH is 130,000. nly the tetrameric
molecule possesses catalytic activity. dhe subunits are expressed in the
following 5 ways:m
È
1
2
ubunits
3
isozyme
4
5
Gctive ite
ëm Most of the amino acid residue in the enzyme are 6 in contact with the
substrate
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ëm Nearly all enzymes are made up of more than 100 amino acid residues,
which give them a mass of greater than 10 kD, and a diameter of more than
25 Å
ëm nly a fraction of the amino acids are involved in the active site formation
2. t is a 3-D entity
ëm E±S complex have an equilibrium constant that range from 10-2 to 10-8 M,
corresponding to free energy of interaction ranging from -3 to -12 kcal/mol
ëm dhese values can be compared with strengths of covalent bonds which are
below -50 to -110 kcal/mol
ëm n all enzymes, the substrate are bound to clefts or crevices from which
water is usually excluded
ëm dhe cleft also has several polar residues that are essential for binding and
catalysis
ëm dhe nonpolar characteristic regions of the cleft enhances the binding ot the
substrate
ëm dhe cleft creates a microenvironment in which certain polar residues acquire
special properties essential for their catalytic role
ëm dhe substrate must have matching shape to fit into the site (ischer¶s model
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ëm dhe active site of some enzymes are not rigid and the active site is thus
modified by the binding of the substrate
ëm dhe active site has a shape complementary to that of the substrate only after
the substrate is bound (Koshland¶s model
ëm dhe side chain groups like ±CH, ±NH2, ±CH2H, etc.. serve as catalytic
groups in the active site
ëm dhe crevice creates a microenvironment in which certain polar residues acquire
special properties essential for catalysis
ëm dhe following figure illustrates the same:
ëm dhere must be at least 3 different points of interaction between the enzyme and
the substrate
ëm dhese interactions can have either a binding or a catalytic function
ëm Binding sites link to specific groups in the substrate, ensuring that the enzyme
and substrate molecules are held in a fixed orientation with respect to each other
with the reacting group or groups in the vicinity of catalytic sites
ëm Consider the following 3-point interaction:
O
ëm Here, sites Gƍƍ and Gƍƍƍ might represent binding sites of Rƍƍ and Rƍƍƍ
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d
ëm Horecker et al., (1962 showed that if the reaction mixture was treated with
sodium borohydride an inactive complex is formed
ëm Upon hydrolysis, ±N±glyceryl lysine was found among the products
ëm rom this, it was concluded that DHG normally binds to the side chain
amino group of a lys residue in the enzyme by a Schiff¶s base (±N=CH±
linkage
ëm dhe substrate in the binding site could be found as follows:
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Ser Chymotrypsin
Ser-195 D D enzyme
(inactivation
Met Chymotrypsin
Met-192 hotooxidation Sulphoxide
product
dyr Carboxypeptidase
dyr-248 odination or nitration nactivation
of benzene
Gsp and Glu epsin J-bromophenacyl nactivation by
bromide ester linkage with
Gsp residue
-carboxylic Lysozyme Gminomethane Loss of enzyme
amino acid sulphonic acid activity
Lys RNase DNB
Lys 1-e41
drp Lysozyme hotooxidation
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ëm dhe ES complex is an intermediate complex and the bonds involved are weak,
non-covalent bonds like hydrogen bonds, ander Waal¶s forces, and
hydrophobic interactions
ëm Sometimes, 2 substrates can bind to an enzyme and such reactions are known as
bisubstrate reactions
ëm Most reactions in the biological system are bisubstrate reactions:
ëm Gll the substrates must bind to the enzyme before any product is released
ëm G ternary complex of the enzyme and both the substrate forms
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( A
(Pyruvate > >> > ( actate ( A
ëm ne or more products are released before all the products are released before all
substrates bind to the enzyme
ëm dhe most important feature is the existence of a substituted enzyme
intermediate, in which the enzyme is temporarily modified
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ëm or example, reactions that shuttle amino groups between amino acids and -
keto acids
ëm Gspartate aminotransferase catalyzes the transfer of an amino group from Gsp
to -ketoglutarate
ëm Gfter Gsp binds to the enzyme, the enzyme removes aspartate¶s amino group to
form the substituted enzyme intermediate
ëm dhe first product GG departs
ëm dhen, the second substrate, -KG binds to the enzyme, accepts the amino group
from the modified enzyme, and is then released as the final product viz.,
glutamine
ormation of t e E Oomplex
ëm dhe site of formation of the ES complex upon the enzyme is known as the
active site
ëm t is made up of several amino acids, that come together as a folding of the 2Y
and 3Y structure of enzymes
ëm So, the active site possess a complete 3-D structure and forms a cleft to accept
the substrate
ëm ormation of the ES complex has been described by 2 models
§m Lock and key model (demplate model by ischer
§m nduced fit model by Koshland
ëm dhese 2 models are elucidated as follows:
ëm dhis model was proposed by ischer which states that the V
6J J V6
66666 6V
ëm dhe active site provides a rigid, pre-shaped template, fitting with the shape
and size of the substrate
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ëm dhe substrate fits into the enzyme as a key fits into a lock; hence this model
is known as the lock and key model
ëm dhis model proposes that the substrate binds with a rigid, preexisting site on
the enzyme
ëm However, this theory cannot explain the change in the enzyme activity in the
presence of allosteric modulators
ëm Due to the restrictive nature of the lock and key model, another model was
proposed by Koshland, known as the induced fit model
ëm dhe important feature of this model is the flexibility of the active site
ëm Gccording to this, the enzyme doesn¶t possess a rigid, preformed structure of
active site to fit in the substrate, but during its binding with the substrate, the
substrate induces a conformational change in the active site of the enzyme to
attain the final shape for binding
ëm Consequently, the enzyme molecule is made to fit completely for the
configuration and active centers of the substrate
ëm Gt the same time, other amino acid residues may become buried in the
interior of the molecule
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ENÀ ME NOMENOGd{E
GLCHL DEHYDRGENGSE
ëm
§m Carbohydrate Carbohydrases
§m roteins roteases
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§m Lipids Lipases
§m Nucleic acids Nucleases
ëm d
§m HydrolysisHydrolases
§m xidationxidases
§m dransaminationdransaminase
§m somerizationsomerases
§m DehydrogenationDehydrogenases
§m hosphorylationhosphorylases
ëm
§m Some enzymes give clue of both the substrates utilized and the type of
reaction catalyzed
§m Examples:
8m L-glutamate dehydrogenase indicates an enzyme catalyzing a
dehydrogenation reaction involving L-glutamic acid
8m Succinate dehydrogenase catalyzes the dehydrogenation of the
substrate succinic acid
ëm
§m G few enzymes are named by adding the suffix ³-ase to the name of
the substance synthesized viz., rhodonase that irreversibily form
hydrocyanic acid and sodium thiosulphate
ëm O
§m Based on their chemical composition, enzymes are classified into
three categories:
8m Enzyme molecule consisting of protein only. Example: pepsin,
trypsin, urease, papain, amylase, etc.
8m Enzyme molecule containing
a protein and a cation. Example:
carbonic
anhydrase (Zn as cation , arginase (Mn2 , tyrosinase
2
(Cu2 , etc.
8m Enzyme molecules containing a protein and a non-protein
organic compound known as prosthetic group. dauber (1950
has subdivided them on the basis of the nature of prosthetic
group involved as follows:
ëm e porphyrin enzymes: Catalase, cyt V peroxidase ,
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ëm Enzymes are broadly classified into 6 classes, according to the type of reaction
catalyzed by them
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d
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ëm Enzymes which catalyze cleavage of bonds by the addition of water are called
hydrolases
ëm dhey catalyze reactions of the type: A X 2 O > >> > X O A
ëm dhey have different classes according to the bond hydrolyzed
ëm or example:
§m 3.1.1.1 Carboxyesterase
§m 3.1.1.2 Gryl esterase
§m 3.2.1.1 -amylase
§m 3.1.3.1 GL
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ëm Lyases cleave a covalent bond of the substrate to convert it into more than one
product, but the reaction does not involve any hydrolysis
ëm dheir action frequently produce a double bond in one of the product
ëm dhey are divided into subclasses according to the atom connected to the bond
ëm dhey generally catalyze the breaking of C±C, C±S, and C±N bonds
ëm Examples:
§m 4.1.1.1 yruvate decarboxylase
§m 4.1.1.22 Histidine decarboxylase
§m 4.3.2.1 Grginosuccinate lyase
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ëm somerases convert their substrates to their isomers by intramolecular
rearrangement
ëm dhey are divided into subclasses according to the type of isomerization
ëm Examples:
§m 5.1.1.1 Glanine raecimase
§m 5.3.1.9 Glucose 6-phosphate isomerase (or phosphohexose isomerase
§m 5.4.2.1 hosphoglycerate mutase
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ëm Ligases are enzymes that catalyze the formation of a bond between 2 substrates
ëm dhey catalyze the formation of bonds between C, , N, and S coupled to
hydrolysis of high energy compounds like Gd
ëm dhey catalyze reactions of the type: Y Gd > >> > 6 Y GD
i or
Y
Gd > >> > 6 Y GM
i
ëm dhey are subdivided into classes according to the atom connected by the new
bond
ëm Examples:
§m 6.1.1.1 dyrosine t-RNG ligase
§m 6.1.1.2 dryptophan t-RNG ligase
§m 6.1.1.3 dhreonine t-RNG ligase
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