BCH 201-INTRODUCTORY BIOCHEMISTRY I- DR SALIU’S LECTURE NOTE

COURSE OUTLINE- Dr SALIU

Structure and chemical properties of amino acids and proteins; classification and reactions of amino
acids. The peptide bond and protein structure. Nature and classification of enzymes, active sites and
specificity of enzyme action. Elementary enzyme kinetics. Factors affecting the velocity of enzyme-
catalyzed reactions. Inhibition and activation of enzymes. Allosteric enzymes and isoenzymes.

AMINO ACIDS AND PROTEINS

Amino acids are the building blocks of proteins. An amino acid is a molecule containing both
amino and carboxyl functional groups. These molecules are particularly important in biochemistry,
where this term refers to alpha-amino acids with the general formula H 2NCHRCOOH, where R is
an organic substituent. In the alpha amino acids, the amino and carboxyl groups are attached to the
same carbon, which is called the α–carbon. The various alpha amino acids differ in which side
chain (R group) is attached to their alpha carbon. They can vary in size from just a hydrogen atom
in glycine through a methyl group in alanine to a large heterocyclic group in tryptophan. An -
amino acid consists of a central carbon atom, called the carbon, linked to an amino group, a
carboxylic acid group, a hydrogen atom, and a distinctive R group. The R group is often referred to
as the side chain. With four different groups connected to the tetrahedral -carbon atom, -amino
acids are chiral; the two mirror-image forms are called the l isomer and the d isomer. Only l amino
acids are constituents of proteins. Proteins are the most abundant biological macromolecules,
occurring in all cells and all parts of cells. Proteins also occur in great variety; thousands of
different kinds, ranging in size from relatively small peptides to huge polymers with molecular
weights in the millions, may be found in a single cell. Proteins are polymers of amino acids, with
each amino acid residue joined to its neighbor by a specific type of covalent bond. (The term
“residue” reflects the loss of the elements of water when one amino acid is joined to another).

CLASSIFICATION OF AMINO ACIDS

The 20 amino acids commonly found as residues in proteins contain an α -carboxyl
group, an α -amino group, and a distinctive R group substituted on the α -carbon atom. The
α -carbon atom of all amino acids except glycine is asymmetric, and thus amino acids can exist
in at least two stereoisomeric forms. Only the L- stereoisomers, with a configuration related to
the absolute configuration of the reference molecule L-glyceraldehyde, are found in
proteins. Other, less common amino acids also occur, either as constituents of proteins (through
modification of common amino acid residues after protein synthesis) or as free metabolites.
Amino acids are classified into five types on the basis of the polarity and charge (at pH 7) of
their R groups.
Nonpolar, Aliphatic R Groups : The R groups in this class of amino acids are nonpolar and
hydrophobic. The side chains of alanine, valine, leucine, and isoleucine tend to cluster together
within proteins, stabilizing protein structure by means of hydrophobic interactions. Glycine has the
simplest structure. Although it is formally nonpolar, its very small side chain makes no real
contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino acids,
has a nonpolar thioether group in its side chain. Proline has an aliphatic side chain with a distinctive
cyclic structure. The secondary amino (imino) group of proline residues is held in a rigid
conformation that reduces the structural flexibility of polypeptide regions containing proline.
Aromatic R Groups: Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are
relatively nonpolar (hydrophobic). All can participate in hydrophobic interactions. The hydroxyl
group of tyrosine can form hydrogen bonds, and it is an important functional group in some
enzymes. Tyrosine and tryptophan are significantly more polar than phenylalanine, because of the
tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. Tryptophan and tyrosine,
and to a much lesser extent phenylalanine, absorb ultraviolet light. This accounts for the
characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a property
exploited by researchers in the characterization of proteins.
Polar, Uncharged R Groups: The R groups of these amino acids are more soluble in water, or
more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups
that form hydrogen bonds with water. This class of amino acids includes serine, threonine, cysteine,
asparagine, and glutamine. The polarity of serine and threonine is contributed by their hydroxyl
groups; that of cysteine by its sulfhydryl group; and that of asparagine and glutamine by their amide
groups. Asparagine and glutamine are the amides of two other amino acids also found in proteins,
aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by
acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called
cystine, in which two cysteine molecules or residues are joined by a disulfide bond. The disulfide-
linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the
structures of many proteins by forming covalent links between parts of a protein molecule or
between two different polypeptide chains.
Positively Charged (Basic) R Groups: The most hydrophilic R groups are those that are either
positively or negatively charged. The amino acids in which the R groups have significant positive
charge at pH 7.0 are lysine, which has a second primary amino group at the ε- position on its
aliphatic chain; arginine, which has a positively charged guanidino group; and histidine, which has
an imidazole group. Histidine is the only common amino acid having an ionizable side chain with a
pKa near neutrality. In many enzyme-catalyzed reactions, a His residue facilitates the reaction by
serving as a proton donor/acceptor.
Negatively Charged (Acidic) R Groups: The two amino acids having R groups with a net
negative charge at pH 7.0 are aspartate and glutamate, each of which has a second carboxyl group.

Uncommon Amino Acids Also Have Important Functions

Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and
5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are
found in collagen, a fibrous protein of connective tissues. 6-NMethyllysine
is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino
acid is α-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other
proteins that bind Ca2+ as part of their biological function. More complex is desmosine, a derivative
of four Lys residues, which is found in the fibrous protein elastin.
Selenocysteine is a special case. This rare amino acid residue is introduced during protein synthesis
rather than created through a postsynthetic modification. It contains selenium rather than the sulfur
of cysteine. Actually derived from serine, selenocysteine is a
constituent of just a few known proteins. Some 300 additional amino acids have been found
in cells. They have a variety of functions but are not constituents of proteins. Ornithine and
citrulline
PROPERTIES OF AMINO ACIDS

(1) Acid-Base Properties

The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and basic
R-groups of the amino acids). As a result of their ionizability, the following ionic equilibrium
reactions may be written:
R-COOH ——> R-COO– + H+

R-NH3+ ——> R-NH2 + H+

The equilibrium reactions, as written, demonstrate that amino acids contain at least two weakly
acidic groups. However, the carboxyl group is a far stronger acid than the amino group. At
physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be
protonated. An amino acid with no ionizable R-group would be electrically neutral at this pH. This
species is termed a zwitterion. Like typical organic acids, the acidic strength of the carboxyl, amino
and ionizable R-groups in amino acids can be defined by the association constant, K a or more
commonly the negative logrithm of Ka, the pKa. The net charge (the algebraic sum of all the
charged groups present) of any amino acid, peptide or protein, will depend upon the pH of the
surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so
too does the net charge. This phenomenon can be observed during the titration of any amino acid or
protein. When the net charge of an amino acid or protein is zero the pH will be equivalent to the
isoelectric point: pI.

(2) Chemical Nature

All peptides and polypeptides are polymers of α-amino acids. There are 20 α-amino acids that are
relevant to the make-up of mammalian proteins. Several other amino acids are found in the body
free or in combined states (i.e. not associated with peptides or proteins). These non-protein
associated amino acids perform specialized functions. Several of the amino acids found in proteins
also serve functions distinct from the formation of peptides and proteins, e.g., tyrosine in the
formation of thyroid hormones or glutamate acting as a neurotransmitter. The α-amino acids in
peptides and proteins (excluding proline) consist of a carboxylic acid (–COOH) and an amino (–
NH2) functional group attached to the same tetrahedral carbon atom. This carbon is the α-carbon.
Distinct R-groups, that distinguish one amino acid from another, also are attached to the alpha-
carbon (except in the case of glycine where the R-group is hydrogen). The fourth substitution on the
tetrahedral α-carbon of amino acids is hydrogen.

(3) Functional Significance of Amino Acid R-Groups

In solution it is the nature of the amino acid R-groups that dictate structure-function relationships of
peptides and proteins. The hydrophobic amino acids will generally be encountered in the interior of
proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are
generally found on the exterior of proteins as well as in the active centers of enzymatically active
proteins. Indeed, it is the very nature of certain amino acid R-groups that allow enzyme reactions to
occur. The imidazole ring of histidine allows it to act as either a proton donor or acceptor at
physiological pH. Hence, it is frequently found in the reactive center of enzymes. Equally important
is the ability of histidines in hemoglobin to buffer the H + ions from carbonic acid ionization in red
blood cells. It is this property of hemoglobin that allows it to exchange O 2 and CO2 at the tissues or
lungs, respectively. The primary alcohol of serine and threonine as well as the thiol (–SH) of
cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the
thiol of cysteine is able to form a disulfide bond with other cysteines:

Cysteine-SH + HS-Cysteine ——> Cysteine-S-S-Cysteine

This simple disulfide is identified as cystine. The formation of disulfide bonds between cysteines
present within proteins is important to the formation of active structural domains in a large number
of proteins. Disulfide bonding between cysteines in different polypeptide chains of oligomeric
proteins plays a crucial role in ordering the structure of complex proteins, e.g. the insulin receptor.

(4) Optical Properties of the Amino Acids

A tetrahedral carbon atom with 4 distinct constituents is said to be chiral. The one amino acid not
exhibiting chirality is glycine since its '"R-group" is a hydrogen atom. Chirality describes the
handedness of a molecule that is observable by the ability of a molecule to rotate the plane of
polarized light either to the right (dextrorotatory) or to the left (levorotatory). All of the amino
acids in proteins exhibit the same absolute steric configuration as L-glyceraldehyde. Therefore, they
are all L-α-amino acids. D-amino acids are never found in proteins, although they exist in nature. D-
amino acids are often found in polypetide antibiotics. The aromatic R-groups in amino acids absorb
ultraviolet light with an absorbance maximum in the range of 280nm. The ability of proteins to
absorb ultraviolet light is predominantly due to the presence of the tryptophan which strongly
absorbs ultraviolet light.

PEPTIDE AND PEPTIDE BOND

Peptide bond formation is a condensation reaction leading to the polymerization of amino acids into
peptides and proteins. Peptides are small consisting of few amino acids. A number of hormones and
neurotransmitters are peptides. Additionally, several antibiotics and antitumor agents are peptides.
Proteins are polypeptides of greatly divergent length. The simplest peptide, a dipeptide, contains a
single peptide bond formed by the condensation of the carboxyl group of one amino acid with the
amino group of the second with the concomitant elimination of water. The presence of the carbonyl
group in a peptide bond allows electron resonance stabilization to occur such that the peptide bond
exhibits rigidity not unlike the typical –C=C– double bond. The peptide bond is, therefore, said to
have partial double-bond character.
PROTEIN
Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of
cells. Proteins also occur in great variety; thousands of different kinds, ranging in size from
relatively small peptides to huge polymers with molecular weights in the millions, may be found in
a single cell. Proteins are linear polymers formed by linking the -carboxyl group of one amino
acid to the -amino group of another amino acid with a peptide bond (also called an amide
bond). The formation of a dipeptide from two amino acids is accompanied by the loss of a water
molecule. The equilibrium of this reaction lies on the side of hydrolysis rather than synthesis.
Hence, the biosynthesis of peptide bonds requires an input of free energy. Nonetheless, peptide
bonds are quite stable kinetically; the lifetime of a peptide bond in aqueous solution in the absence
of a catalyst approaches 1000 years.
A series of amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit
in a polypeptide is called a residue. A polypeptide chain has polarity because its ends are different,
with an -amino group at one end and an -carboxyl group at the other. By convention, the
amino end is taken to be the beginning of a polypeptide chain, and so the sequence of amino acids
in a polypeptide chain is written starting with the aminoterminal residue. Thus, in the pentapeptide
Tyr-Gly-Gly-Phe-Leu (YGGFL), phenylalanine is the amino-terminal (N-terminal) residue and
leucine is the carboxyl-terminal (C-terminal) residue (Figure 3.19). Leu-Phe-Gly-Gly-Tyr
(LFGGY) is a different pentapeptide, with different chemical properties.
A polypeptide chain consists of a regularly repeating part, called the main chain or backbone, and a
variable part, comprising the distinctive side chains. The polypeptide backbone is rich in hydrogen-
bonding potential. Each residue contains a carbonyl group, which is a good hydrogen-bond acceptor
and, with the exception of proline, an NH group, which is a good hydrogen-bond donor. These
groups interact with each other and with functional groups from side chains to stabilize particular
structures, as will be discussed in detail. Most natural polypeptide chains contain between 50 and
2000 amino acid residues and are commonly referred to as proteins. Peptides made of small
numbers of amino acids are called oligopeptides or simply peptides. The mean
molecular weight of an amino acid residue is about 110, and so the molecular weights of most
proteins are between 5500 and 220,000. We can also refer to the mass of a protein, which is
expressed in units of daltons; one dalton is equal to one atomic mass unit. A protein with a
molecular weight of 50,000 has a mass of 50,000 daltons, or 50 kd (kilodaltons).

Proteins can be broken down (hydrolyzed) to their constituent amino acids by a variety of methods,
and the earliest studies of proteins naturally focused on the free amino acids derived from them.
Twenty different amino acids are commonly found in proteins. All 20 of the common amino acids
are -amino acids. They have a carboxyl group and an amino group bonded to the same carbon
atom (the - carbon). They differ from each other in their side chains, or R groups, which vary in
structure, size, and electric charge, and which influence the solubility of the amino acids in water. In
addition to these 20 amino acids there are many less common ones. Some are residues modified
after a protein has been synthesized; others are amino acids present in living organisms but not as
constituents of proteins. The common amino acids of proteins have been assigned three-letter
abbreviations and one-letter symbols, which are used as shorthand to indicate the composition and
sequence of amino acids polymerized in proteins. Two conventions are used to identify the carbons
in an amino acid—a practice that can be confusing. The additional carbons in an R group are
commonly designated ß, γ, d, € and so forth, proceeding out from the -carbon. For most other
organic molecules, carbon atoms are simply numbered from one end, giving highest priority (C-1)
to the carbon with the substituent containing the atom of highest atomic number. Within this latter
convention, the carboxyl carbon of an amino acid would be C-1 and the - carbon would be C-2.
In some cases, such as amino acids with heterocyclic R groups, the Greek lettering system is
ambiguous and the numbering convention is therefore used.
For all the common amino acids except glycine, the - carbon is bonded to four different groups: a
carboxyl group, an amino group, an R group, and a hydrogen atom in glycine, the R group is
another hydrogenatom). The -carbon atom is thus a chiral center. Because of the tetrahedral
arrangement of the bonding orbitals around the -carbon atom, the four different groups can
occupy two unique spatial arrangements, and thus amino acids have two possible stereoisomers.
Since they are nonsuperimposable mirror images of each other, the two forms represent a class of
stereoisomers called enantiomers. All molecules with a chiral center are also optically active—
that is, they rotate plane-polarized light.

PROTEIN STRUCTURE AND FUNCTION

The three-dimensional structure of a protein that is often key to its enzyme function. The structure
of a protein is characterized in four ways: The primary structure is the order of the different amino
acids in a protein chain, whereas the secondary structure consists of the geometry of chain segments
in forms such as helices or sheets. The tertiary structure describes how a protein folds in on itself;
the quaternary structure of a protein describes how different protein chains hook up with each other

Proteins play crucial roles in almost every biological process. They are responsible in one form or
another for a variety of physiological functions including:

(1) Enzymatic catalysis - almost all biological reactions are enzyme catalyzed. Enzymes are known
to increase the rate of a biological reaction by a factor of 10 to the 6th power! There are several
thousand enzymes which have been identified to date.
(2) Binding, transport and storage - small molecules are often carried by proteins in the
physiological setting (for example, the protein hemoglobin is responsible for the transport of
oxygen to tissues). Many drug molecules are partially bound to serum albumins in the plasma.
(3) Molecular switching - conformational changes in response to pH or ligand binding can be used
to control cellular processes
(4) Coordinated motion - muscle is mostly protein, and muscle contraction is mediated by the
sliding motion of two protein filaments, actin and myosin.
(5) Structural support - skin and bone are strengthened by the protein collagen.
(6) Immune protection - antibodies are protein structures that are responsible for reacting with
specific foreign substances in the body.
(7) Generation and transmission of nerve impulses - some amino acids act as neurotransmitters,
which transmit electrical signals from one nerve cell to another. In addition, receptors for
neurotransmitters, drugs, etc. are protein in nature. An example of this is the acetylcholine
receptor, which is a protein structure that is embedded in postsynaptic neurons.
(8) Control of growth and differentiation - proteins can be critical to the control of growth, cell
differentiation and expression of DNA. For example, repressor proteins may bind to specific
segments of DNA, preventing expression and thus the formation of the product of that DNA
segment. Also, many hormones and growth factors that regulate cell function, such as insulin or
thyroid stimulating hormone are proteins.

Proteins have a total of four levels of structure, as defined below:

(1) Primary structure - this term refers to the amino acid sequence of a protein, including cystines
that are formed during crosslinking. Sequence can dictate three dimensional structure, since amino
acid residues need to be in a specific order to foster proper protein folding, and since disulfides
must be formed from properly positioned cysteines to afford an active protein. An example of
primary structure is the hypertensive octapeptide angiotensin II, which has the sequence ASP-
ARG-VAL-TYR-ILE-HIS-PRO-PHE.

(2) Secondary structure - this term refers to the arrangement of amino acids that are close together
in a chain. Examples of secondary structures are helices and pleated sheets. An alpha helix is a
tightly coiled, rodlike structure which has an average of 3.6 amino acids per turn. The helix is
stabilized by hydrogen bonding between the backbone carbonyl of one amino acid and the
backbone NH of the amino acid four residues away. All main chain amino and carboxyl groups are
hydrogen bonded, and the R groups stick out from the structure in a spiral arrangement. As seen in
the table below, there are several types of alpha helix that arise from the degree of hydrogen
bonding in the helix.
Another type of secondary structure, the beta pleated sheet is composed of two or more straight
chains that are hydrogen bonded side by side. If the amino termini are on the same end of each
chain, the sheet is termed parallel, and if the chains run in the opposite direction (amino termini on
opposite ends), the sheet is termed antiparallel (see below left). All of the amides are hydrogen
bonded except those on the outer strands. Pleated sheets may be formed from a single chain if it
contains a beta turn, which forms a hairpin loop structure. Often a proline can be found in a beta
turn, since it places a "kink" in the chain. When the beta sheet curves around itself and the outer
edges on either side hydrogen bond to one another, it forms a structure called a beta barrel, which is
a common structural motif in proteins.

(3) Tertiary structure - tertiary structure refers to the arrangement of amino acids that are far apart
in the chain. Each protein ultimately folds into a three dimensional shape with a distinct inside
and outside. The interior of a protein molecule contains a preponderance of hydrophobic amino
acids, which tend to cluster and exclude water. The core is stabilized by Van der Waals forces and
hydrophobic bonding. By contrast, the exterior of a protein molecule is largely composed of
hydrophilic amino acids, which are charged or able to H-bond with water. This allows a protein to
have greater water solubility. A protein will spontaneously fold to preserve the relationships
outlined above.

(4) Quaternary structure - protein chains can associate with other chains to form dimers, trimers,
and other higher orders of oligomers. Generally they contain between 2 and 6 subunits which
may be chains with the same sequence (homodimers) or different chains (heterodimers).
Nature and classification of enzymes, active sites and specificity of enzyme action.

INTRODUCTION TO ENZYMES
Enzymes are special kinds of proteins that consist of long chains of amino acids bound together by
peptide bonds. They are found in all living cells, performing specifi c functions, i.e., controlling the
metabolic processes, converting nutrients into energy, breaking down food materials, etc. For
example,
the enzymes pepsin, trypsin, and peptidases break down proteins into amino acids, the lipases
split fats into glycerol and fatty acids, and the amylases break down starch into simple sugars.
Enzymes are some of the proteins that are made by cells from amino acids. These are linked
together by strong, covalent bonds, which are referred to as “peptide bonds.” Thus each enzyme
(as a protein) has a different amino acid sequence called “primary structure
A coenzyme or metal ion that is very tightly or evencovalently bound to the enzyme protein is
called a prosthetic group. Acomplete, catalytically active enzyme together with its bound
coenzymeand/or metal ions is called a holoenzyme. The protein part of such an enzymeis called the
apoenzyme or apoprotein.
ENZYME CHARACTERISTICS
In general, living systems control their activities through the aid of enzymes, which are protein
molecules,
i.e., biological catalysts with the following three main characteristics.7 First, the main function
of an enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million
times faster than they would in the absence of an enzyme. Second, most enzymes act specifi cally
with only one reactant called “substrate” in order to produce a product (Figure 4.2). The third
characteristic
is that enzymes are regulated from a state of low activity to high activity and vice versa.
form of the enzyme carboxypeptidase (shown in Figure 4.2) hydrolyzes a peptide at the carboxyl or
the C terminal end of the chain.
Some of the characteristics of enzymes are the following:
1. High activity
2. Selectivity
3. Regiospecifi city
4. Stereo-specifi city
5. Controllability
6. Environmentally friendly
High activity. Enzymes can increase the rate of a reaction millions of times by lowering the
activation
energy of the reaction like conventional chemical catalysts. The maximum rate of conversion of
a substrate to a product by a molecule of an enzyme is known as the “turnover number” (Kcat). For
example, the Kcat for catalase, which catalyzes the conversion of hydrogen peroxide to O2 and
H2O,
is approximately 600,000 molecules per second per molecule of enzyme.
Selectivity. The catalytic active site of an enzyme, which is the result of folding of the polypeptide
chain, fi ts only to a small (specifi c) number of substrates for the conversion to products. Since an
enzyme acts only on one compound of a complex mixture, it can control a reaction independently
of many others occurring in the complex. Minor changes, like increasing the temperature, in the
structure of the substrate may cause the enzyme to be unable to recognize the substrate and convert
it to corresponding products.
Regiospecifi city. An enzyme can detect differences in the spatial arrangement of atoms in a
compound.
For example, monoxygenase enzymes oxidize methyl cyclohexane, but different enzymes
are found to oxidize either the methyl substitute or the methylene ring group. This has potential
application in the production of synthetic fragrances and fl avors.
Stereospecifi city. Enzymes are highly stereospecifi c in the choice of substrate or in the formed
product.
Their selectivity for the substrate usually extends to discernment between different forms (chiral) of
the substrate molecule (d or l forms). For example, bacterial glucose oxidase will use only d-
glucose.
Controllability. The catalysis of reactions by enzymes can be controlled by the amount of substrate
and by other factors (temperature, pH, etc.). An enzyme is active (switched on) in the presence of a
correct
amount of the substrate and inactive (turned off) when the substrate is not available. For example,
the response of certain bacteria to the presence of lactose induces the formation of β-galactosidase
(lactase), which hydrolyses lactose to glucose and galactose used as food by organisms.
Environmental factors. Reactions catalyzed by enzymes (biocatalysts with high specifi city) are less
environmentally polluting due to the lower by-product produced. The synthesized products can
be classifi ed as “natural” when substrates are naturally derived, and they are valuable to the food
industry as they are not chemically manufactured.
CLASSIFICATION OF ENZYMES
Enzyme classifi cation is primarily based on the recommendations of the Nomenclature Committee
of the International Union of Biochemistry and Molecular Biology (IUBMB)12, and it describes
each
type of characterized enzyme for which an EC (Enzyme Commission) number has been provided.
EC classes defi ne enzyme function based on the reaction, which is catalyzed by the enzyme. The
classifi cation scheme is hierarchical, with four levels. There are six broad categories of function at
the
top of this hierarchy and about 3500 specifi c reaction types at the bottom. EC classes are expressed
as a string of four numbers separated by periods. EC class strings with fewer than four numbers
refer
to an internal node in the tree, implicitly including all of the subclasses and leaves below it. The
numbers
specify a path down the hierarchy, with the leftmost number identifying the highest level.
4.1.5.1 Oxidoreductases
These enzymes catalyze oxidation–reduction reactions in which hydrogen or oxygen atoms or
electrons
are transferred between molecules. This large class includes dehydrogenases (hydride transfer),
oxidases (electron transfer to molecular oxygen), oxygenases (oxygen transfer from molecular
oxygen), and peroxidases (electron transfer to peroxide). An example is shown in Figure 4.4, with
the glucose oxidase (EC 1.1.3.4; systematic name, β-d-glucose-oxygen 1-oxidoreductase) reaction
and its products.
4.1.5.2 Transferases
These enzymes catalyze the transfer of an atom or group of atoms (e.g., acyl-, alkyl-, and
glycosyl-),
from one molecular and/or functional groups to another, but excluding such transfers as are classifi
ed
in the other groups (e.g., oxidoreductases and hydrolases). An example is shown in Figure 4.5, with
the aspartate aminotransferase (EC 2.6.1.1; systematic name, l-aspartate: 2-oxoglutarate
aminotransferase;
also called glutamic-oxaloacetic transaminase or simply GOT) reaction and its product.
4.1.5.3 Hydrolases
These involve hydrolytic reactions and their reversal (degradation of H2O to OH− and H+
products).
This is presently the most commonly encountered class of enzymes within the fi eld of enzyme
technology
and includes the esterases, glycosidases, lipases, and proteases. An example of the chymosin
(EC 3.4.23.4; no systematic name declared; also called rennin) reaction and its product are shown
in Figure 4.6.
4.1.5.4 Lyases
Lyases involve elimination reactions in which a group of atoms is removed from the substrate.
These catalytic reactions require the addition of groups to a double bond or the formation of a
double bond (e.g., C=C, C=N, C=O). This includes the aldolases, decarboxylases, dehydratases,
and some pectinases but does not include hydrolases. An example is the histidine ammonia-lyase
(EC 4.3.1.3; systematic name, l-histidine ammonia-lyase, which is also called histidase) reaction
with its products, shown in Figure 4.7.
4.1.5.5 Isomerases
Isomerases catalyze molecular isomerizations and include the epimerases, racemases, and
intramolecular transferases. An example is shown in Figure 4.8, with the xylose isomerase (EC
5.3.1.5; systematic name, d-xylose ketol-isomerase; commonly called glucose isomerase)
transformation of
α-d-glucopyranose to α-d-fructofuranose.

4.1.5.6 Ligases
Ligases catalyze the condensation of two molecules together with the cleavage of ATP or another
pyrophosphate bond. They, also known as synthetases, form a relatively small group of enzymes,
which involve the formation of a covalent bond joining two molecules together, coupled with the
hydrolysis of a nucleoside triphosphate. An example is the glutathione synthase (EC 6.3.2.3;
systematic
name, α-l-glutamyl-l-cysteine:glycine ligase (ADP-forming), which, also, called glutathione
synthetase) reaction, shown in Figure 4.9 with its products.
Each enzyme category (e.g., EC 1) is divided into subclasses (e.g., EC 1.2) and its subclass is
subdivided
into sub-subclasses (e.g., EC 1.2.2). The last (fourth) number of the IUBMB nomenclature
refers to the particular enzyme (e.g., EC 1.2.2.3). For example, the enzyme EC 1.10.3.2 (laccase)
represents an oxidoreductase enzyme (EC 1) that acts on diphenols and related substances as donors
(EC 1.10), with oxygen as the acceptor (EC 1.10.3). Table 4.2 shows the six EC enzyme classes
with their functionalities.
In industry, the two major groups of enzyme categories, which are being used, are (1) the
oxidoreductases, for example, catalase and glucose oxidase and (2) the hydrolases, for example,
amylases, proteases, cellulase, invertase, etc. Transferases, lyases, and ligases are not used
extensively, while some isomerases have been extremely useful in the industries (production of
syrups). In industry, there are the following three main ways in which enzymes can be used as (1)
soluble (fungal α-amylase—baking); (2) immobilized (bacterial glucose isomerase—syrups, e.g.,
HFCS); and (3) nonproliferating whole cells (Pseudomonas putida—adipic acid). Soluble and
immobilized
enzymes are intracellular or extracellular, while nonproliferating whole cells are nondividing cells.
Examples of these enzymes are given bellow. The soluble enzymes are not used in continuous
processes
but in batch-type reactors because they are sacrifi ced after the reaction is completed and are
not economically justifi ed for recovery. These enzymes, usually, diffuse in the substrate and need
to
be rinsed or inactivated to control the reaction.13
The immobilized enzymes are those enzymes that retain their catalytic activity, while their
movement is physically restricted or localized in a defi ned space.14 For example, an immobilized
enzyme, within a matrix, should be able to diffuse the substrate and product in and out of the
matrix, freely.15 In other words, an immobilized enzyme is retained in the reactor while the desired
product freely moves out, allowing a continual production of the desired product. Researchers,
enzymologists, and biotechnologists are very much interested in immobilized enzymes and more
specifi cally soluble enzymes as heterogeneous catalysts. Nonproliferating whole cells are those that
are metabolically active but are starved of an essential nutrient for growth. They perform
multisequence reactions.16,17
TABLE 4.2
The EC Enzyme Classes with Their Activities
EC 1 Oxidoreductases
EC 1.1 Acting on CH-OH groups of donors
EC 1.2 Acting on aldehyde or oxo groups of donors
EC 1.3 Acting on CH-CH groups of donors
EC 1.4 Acting on CH-NH2 groups of donors
EC 1.5 Acting on CH-NH groups of donors
EC 1.6 Acting on NADH or NADPH
EC 1.7 Acting on other nitrogenous compounds as donors
EC 1.8 Acting on sulfur groups of donors
EC 1.9 Acting on hemi groups of donors
EC 1.10 Acting on diphenols and related substances as donors
EC 1.11 Acting on a peroxide as acceptors
EC 1.12 Acting on hydrogen as donors
EC 1.13 Acting on single donors, incorporation of molecular oxygen (oxygenases)
EC 1.14 Acting on paired donors, incorporation or reduction of molecular oxygen
EC 1.15 Acting on superoxide radicals as acceptors
EC 1.16 Oxidizing metal ions
EC 1.17 Acting on CH or CH2 groups
EC 1.18 Acting on iron-sulfur proteins as donors
EC 1.19 Acting on reduction fl avodoxin as donors
EC 1.20 Acting on phosphorus or arsenic in donors
EC 1.21 Acting on X-H and Y-H to form X-Y bonds
EC 1.97 Other oxidoreductases
EC 2 Transferases
EC 2.1 Transferring one-carbon groups
EC 2.2 Transferring aldehyde or ketonic groups
EC 2.3 Acyltransferases
EC 2.4 Glycosyltransferases
EC 2.5 Transferring alkyl or aryl groups, other than methyl groups
EC 2.6 Transferring nitrogenous groups
EC 2.7 Transferring phosphorus-containing groups
EC 2.8 Transferring sulfur-containing groups
EC 2.9 Transferring selenium-containing groups
EC 3 Hydrolases
EC 3.1 Acting on ester bonds
EC 3.2 Glycosidases
EC 3.3 Acting on ether bonds
EC 3.4 Acting on peptide bonds (peptidases)
EC 3.5 Acting on carbon bonds, other than peptide bonds
EC 3.6 Acting on acid anhydrides
EC 3.7 Acting on carbon-carbon bonds
EC 3.8 Acting on halide bonds
EC 3.9 Acting on phosphorus-nitrogen bonds
EC 3.10 Acting on sulfur-nitrogen bonds
EC 3.11 Acting on carbon-sulfur bonds
EC 3.12 Acting on sulfur-sulfur bonds
EC 3.13 Acting on carbon-sulfur bonds
(The EC Enzyme Classes with Their Activities
EC 4 Lyases
EC 4.1 Carbon-carbon lyases
EC 4.2 Carbon-oxygen lyases
EC 4.3 Carbon-nitrogen lyases
EC 4.4 Carbon-sulfur lyases
EC 4.5 Carbon-halide lyases
EC 4.6 Phosphorus-oxygen lyases
EC 4.99 Other lyases
EC 5 Isomerases
EC 5.1 Recamases and epimerases
EC 5.2 cis–trans-Isomerases
EC 5.3 Intramolecular isomerases
EC 5.4 Intramolecular transferases (mutases)
EC 5.5 Intramolecular lyases
EC 5.99 Other isomerases
EC 6 Ligases
EC 6.1 Forming carbon-oxygen bonds
EC 6.2 Forming carbon-sulfur bonds
EC 6.3 Forming carbon-nitrogen bonds
EC 6.4 Forming carbon-carbon bonds
EC 6.5 Forming phosphoric ester bonds
EC 6.6 Forming nitrogen-metal bonds
Sources: NC-IUBMB, Enzyme nomenclature: Recommendations of the Nomenclature
Committee of the International Union of Biochemistry and Molecular Biology
on the nomenclature and classifi cation of enzyme-catalyzed reactions, available
online at www.chem.qmul.ac.uk/iubmb/enzyme/
How Enzymes Work
The enzymatic catalysis of reactions is essential to living systems. Under biologically relevant
conditions, uncatalyzed reactions tend to be slow—most biological molecules are quite stable in the
neutral-pH, mild-temperature, aqueous environment inside cells. Furthermore, many common
chemical processes are unfavorable or unlikely in the cellular environment, such as the transient
formation of unstable charged intermediates or the collision of twoor more molecules in the precise
orientation required for reaction. Reactions required to digest food, send nerve signals, or contract a
muscle simply do not occur at a useful rate without catalysis. An enzyme circumvents these
problems by providing a specificenvironment in which a given reaction can occur more rapidly. The
distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a
pocket on the enzyme called the active site (Fig. 6-1). The molecule that is bound in the active site
and acted upon by the enzyme is called the substrate. The surface of the active site is lined with
amino acid residues with substituent groups that bind the substrate and catalyze its chemical
transformation. Often, the active site encloses a substrate, sequestering it completely from solution.
The enzyme-substrate complex, an entity first proposed by Charles-Adolphe Wurtz in 1880, is
central to the action of enzymes. It is also the starting point for mathematical treatments that define
the kinetic behavior of enzyme-catalyzed reactions and for theoretical descriptions of enzyme
mechanisms. The enzyme active site is structured so that some of these weak interactions occur
preferentially in the reaction transition state, thus stabilizing the transition state.

ELEMENTARY ENZYME KINETICS

Enzymes are known as protein catalysts that, like all other catalysts, speed up the rate of a chemical
reaction without being used up in the process. A catalyst is a substance that increases the
rate of a reaction without modifying the overall standard Gibbs energy change in the reaction
(Figure 4.10). An uncatalyzed reaction requires higher activation energy than does a catalyzed
reaction.19 This defi nition is equivalent to the statement that the catalyst does not appear in the
stoichiometric expression of the complete reaction. Catalysts are said to exert a catalytic action,
and a reaction in which a catalyst is involved is called a catalyzed reaction.
Kinetic equations are commonly expressed in terms of the amount-of-substance concentrations
of the chemical species involved. The amount-of-substance concentration is the amount of
substance
(for which the SI unit is the mole, symbol mol) divided by the volume. As it is the only kind of
concentration
commonly used in biochemistry it is usually abbreviated to concentration and this shorter
form will be used in the remainder of this document without further discussion. The unit almost
invariably used for concentration is mol dm−3, which is alternatively written as mol L−1, mol l−1,
or simply ΚMΚ (molar).20,21
Added substances sometimes increase or decrease the rate of an enzyme-catalyzed reaction
without interacting with the enzyme itself. These substances may interact with substrates or
with modifi ers or effectors that are already present in the system. Such substances are referred
to as activators or inhibitors, but not as enzyme activators, enzyme inhibitors, modifi ers, or
effectors.22
An inhibitor is a substance that diminishes the rate of a chemical reaction and the process is
called inhibition. In enzyme-catalyzed reactions an inhibitor frequently acts by binding to the
enzyme, in which case it may be called an enzyme inhibitor. An activator is a substance, other than
the catalyst or one of the substrates, that increases the rate of a catalyzed reaction. An
activator of an enzyme-catalyzed reaction may be called an enzyme activator if it acts by binding
to the enzyme.
A typical overall enzyme-catalyzed reaction involving a single substrate and a single product
may be written as23
Enzyme + substrate_(enzyme − substrate complex)→enzyme + product
++
−
+ ________ ⎯⎯→ + 1 2
1
E S ES P E k k
k
(4.1)
where k+1, k−1, and k+2 are the respective rate constants, typically having values of 105–108 M−1
s−1,
1–104 s−1, and 1–105 s−1 respectively.
The rate at which an enzyme works is infl uenced by the following:24
1. The concentration of substrate molecules (the more of them available, the quicker the
enzyme molecules collide and bind with them). The concentration of substrate is designated
[S] and is expressed in the unit of “molarity.”
2. The temperature. As the temperature rises, molecular motion—and hence collisions
between enzyme and substrate—speed up. This cannot be infi nite since enzymes are
proteins that have an upper limit beyond which the enzyme becomes denatured and
ineffective.
3. The presence of inhibitors. They are classifi ed in the following two categories:
a. Competitive inhibitors are molecules that bind to the same site as the substrate preventing
the substrate from binding—but are not changed by the enzyme.
b. Noncompetitive inhibitors are molecules that bind to some other site on the enzyme
reducing its catalytic power.
4. pH. The conformation of a protein is infl uenced by pH and as enzyme activity is crucially
dependent on its conformation, its activity is likewise affected.
The study of the rate at which an enzyme works is called enzyme kinetics.25 Following that,
enzyme kinetics will be examined as a function of the concentration of the substrate available to
the enzyme.
First let us set up a series of tubes containing graded concentrations of substrate, [S].26 At time
zero, we add a fi xed amount of the enzyme preparation. Over the next few minutes, we measure the
concentration of the product formed. If the product absorbs light, we can easily do this in a
spectrophotometer.
Early in the run, when the amount of substrate is in substantial excess to the amount
of enzyme, the rate we observe is the initial velocity of Vi. By plotting Vi as a function of [S], the
following are observed (Figure 4.11).
• At low values of [S], the initial velocity, Vi, rises almost linearly with increasing [S].
• But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).
• The asymptote represents the maximum velocity of the reaction, designated Vmax.
• The substrate concentration that produces a Vi that is one-half of Vmax is designated the
Michaelis–Menten constant, Km.
Km is an inverse measure of the affi nity or strength of binding between the enzyme and its
substrate.
The lower the Km, the greater the affi nity (so the lower the concentration of substrate needed to
achieve a given rate).
INHIBITION
The inhibition is divided into the following two categories:27
1. Competitive, when the substrate and inhibitor compete for binding to the same active site
2. Noncompetitive, when the inhibitor binds somewhere else on the enzyme molecule reducing
its effi ciency
The distinction can be determined by plotting enzyme activity with and without the inhibitor present
Competitive Inhibition
In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the
same
velocities that were reached in its absence. So while Vmax can still be reached if suffi cient
substrate is
available, one-half Vmax requires a higher [S] than before and thus Km is larger (Figure 4.13).
4.2.1.2 Noncompetitive Inhibition
With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are not
counted so the following (Figure 4.14) are displayed by the Lineweaver–Burk plot.
1. Enzyme rate (velocity) is reduced for all values of [S], including
2. Vmax and one-half Vmax, but
3. Km remains unchanged because the active site of those enzyme molecules that have not
been inhibited is unchanged.
ALLOSTERIC ENZYMES
Allosteric Enzymes Undergo Conformational Changes in Response to Modulator Binding.
Allosteric proteins are those having “other shapes” orconformations induced by the binding of
modulators. The same conceptapplies to certain regulatory enzymes, as conformational changes
induced byone or more modulators interconvert more-active and less-active forms of theenzyme.
The modulators for allosteric enzymes may be inhibitory orstimulatory. Often the modulator is the
substrate itself; regulation in whichsubstrate and modulator are identical is referred to as
homotropic. The effectis similar to that of O2 binding to hemoglobin (Chapter 5): binding of the
ligand—or substrate, in the case of enzymes—causes conformational changesthat affect the
subsequent activity of other sites on the protein. In most cases,the conformational change converts a
relatively inactive conformation (oftenreferred to as a T state) to a more active conformation (an R
state). When themodulator is a molecule other than the substrate, the enzyme is said to
beheterotropic. Note that allosteric modulators should not be confused withuncompetitive and
mixed inhibitors. Although the latter bind at a second siteon the enzyme, they do not necessarily
mediate conformational changes between active and inactive forms, and the kinetic effects are
distinct. The properties of allosteric enzymes are significantly different from thoseof simple
nonregulatory enzymes. Some of the differences are structural. In addition to active sites, allosteric
enzymes generally have one or moreregulatory, or allosteric, sites for binding the modulator (Fig. 6-
33). Just as anenzyme’s active site is specific for its substrate, each regulatory site isspecific for its
modulator. Enzymes with several modulators generally havedifferent specific binding sites for each.
In homotropic enzymes, the activesite and regulatory site are the same.FIGURE 6-33 Subunit
interactions in an allosteric enzyme, andinteractions with inhibitors and activators. In many
allosteric enzymes, the substrate-binding site and the modulator-binding site(s) are on different
subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding ofthe positive
(stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the
catalytic subunit through a conformational change. This change renders the catalytic subunit active
and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from
the regulatory subunit, the enzyme reverts to its inactive or less active form. Allosteric enzymes are
typically larger and more complex than nonallosteric enzymes, with two or more subunits. A classic
example is aspartate transcarbamoylase (often abbreviated ATCase), which catalyzes a nearly step
in the biosynthesis of pyrimidine nucleotides, the reaction ofcarbamoyl phosphate and aspartate to
form carbamoyl aspartate: ATCase has 12 polypeptide chains organized into 6 catalytic
subunits(organized as 2 trimeric complexes) and 6 regulatory subunits (organized as 3dimeric
complexes). Figure 6-34 shows the quaternary structure of thisenzyme, deduced from x-ray
analysis. The enzyme exhibits allostericbehavior as detailed below, as the catalytic subunits
function cooperatively.The regulatory subunits have binding sites for ATP and CTP, which
functionas positive and negative regulators, respectively. CTP is one of the endproducts of the
pathway, and negative regulation by CTP serves to limitATCase action under conditions when CTP
is abundant. On the other hand,high concentrations of ATP indicate that cellular metabolism is
robust, thecell is growing, and additional pyrimidine nucleotides may be needed tosupport RNA
transcription and DNA replication.
ISOENZYMES

Isoenzymes or isozymes are proteins which have different molecular and physicochemical
properties, but have identical catalytic activity. They generally occur in branched metabolic
pathways in which several end-products are synthesized from a common precursor molecule.

A hypothetical branched pathway in which three different end-products, X, Y and Z, are

synthesized from a common precursor A is shown:

In the above hypothetical pathway, if a single enzyme catalyse the first step i.e. conversion of A to
B, then over-production of any one end-product, X or Y or Z, would stop conversion of A to B, and
consequently, production of all the end-products would stop.

This is not desirable for the cell because the other two end-products might be essential. If, on the
other hand, three different enzymes exist catalyzing conversion of A to B, which are individually
inhibited by feedback mechanism by the end-products, X, Y and Z, then inhibition of any one
enzyme by its end-product would not affect the biosynthesis of other two products. Such different
enzymes catalyzing the same reaction are isozymes. They are also called parallel enzymes.
Isoenzymes play a crucial role in the regulation of branched pathways.