CH6705 Biochemical Engineering ASSIGNMENT-II 2016-2017

Part – A
1. What is an Enzyme? (Nov 2013)
A substance produced by a living organism which acts as a catalyst to bring about a specific
biochemical reaction.

2. What are the methods available for immobilization of whole cells? (May 2014)
i) Entrapment techniques (ii) Adsorption Techniques
(iii) Selective binding of cells by immobilized macromolecules.
(iv) Covalent bonding of cell to support.

3. Mention the methods used in evaluating Michealis – Menten parameters. (Nov

2012)
Line weaver Burk plot
Langmuir plot method
Eadie – Hofstee plot method.

4. Mention the various approaches used in deriving the reaction rate for any enzyme
catalysed reaction (Nov 2013)
(i) Michealis – Menten approach. The equation is given by

(ii) Brigg’s – Haldane approach.

5. Compare the role of enzymes and cells in the manufacture of a biochemical

product.
(Nov 2014) What is a cofactor? (May 2013)
Each reaction in the cell is catalyzed by its own, specific enzyme to synthesis a biochemical
product. The enzyme catalysts regulate the structure and function of cells and organisms.

Cofactor
It is a non – protein compound which combines with an inactive protein to give a
catalytically active complex.

6. Explain competitive inhibition. (May 2015)

Competitive inhibition is a form of enzyme inhibition where binding of the inhibitor to
the active site on the enzyme prevents binding of the substrate and vice versa.
Most competitive inhibitors function by binding reversibly to the active site of the
enzyme.

7. Define units of enzyme activity. (May 2015)

The enzyme unit (U) is a unit for the amount of a particular enzyme. One U is defined as
the amount of the enzyme that produces a certain amount of enzymatic activity, that is,
the amount that catalyzes the conversion of 1 micro mole of substrate per minute.

8. What do you understand by sterilization? (Nov 2011, May 2015)

An important aspect to be taken care of in bioprocesses unlike chemical reactions, because
presence of environmental bacteria and microorganisms consume the nutrients from the
media, making the process more difficult with the cultivation of plant or animal cells
because their growth rates are much slower than those of environmental bacteria or molds.
PART – B

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1. i)How to Enzymes are classified?

Table 3.1 Enzyme Commission classification system for enzymes (class names,
Enzyme Commission type numbers, and type of reactions catalyzed) A. L. Lehninger,
Biochemistsry, 2d ed., table 8-1, Worth publishers, Inc., New York, 1975.

1. Oxidoreductases (oxidation-reduction reactions)

|
1.1 Acting on  CH OH
|
1.2 Action on  C  O
1.3 Action on CH  CH 
|
1.4 Action on  CH NH2
|
1.5 Action on  CH NH 
1.6 Action on NADH;
………………………….
2. Transferases (transfer of functional groups)
2.1 One-carbon groups
2.2 Aldehydic or ketonic groups
2.3 Acyl groups
2.4 Glycosyl groups
…………………………
2.7 Phosphate groups
2.8 S-containing groups
3. Hydrolases (hydrolysis reactions)
3.1 Esters
3.2 Glycosidic bonds
…………………………
3.4 Peptide bonds
3.5 Other C-N bonds
3.6 Acid anhydrides
………………………..

4. Lyases ( addition to double bonds)

| |
4.1  C  C
|
4.2  C  O
|
4.3  C  N 
………………………….

5. Isomerases (isomerization reactions)

5.1 Racemases
………………………….

6. Ligases (formation of bonds with ATP cleavage)

6.1 C-O
6.2 C-S
6.3 C-N
6.4 C-C
………..

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ii) Explain the Immobilized Enzyme Technology?

Immobilization of an enzyme means that it has been confined or localized so that it can be
reused continuously. There are several reasons why immobilization may be desirable: for
processing with isolated enzymes, an immobilized form can be retained in the reactor. With
enzymes, an immobilized form can be retained in the reactor. With enzymes in solution, on the
other hand, some enzymes will leave the reactor with the final product. Not only must new
enzymes be introduced to replace the lost ones, but enzymes in the product may be undesirable
impurities which must be removed. Immobilized enzymes may retain their activity longer than
those in solution (figure). Finally, an immobilized enzyme may be fixed in position near other
enzymes participating in a catalytic sequence, thereby increasing the catalyst efficiency for the
multistep conversion.
These characteristics make immobilized enzymes attractive if a very large throughput of
substrate is required and/or the enzymes involved are expensive. Moreover, the ability to confine
an enzyme in a well-defined, predetermined space provides opportunities for applications unique
to immobilized enzymes. In the next section we examine methods of enzyme immobilization. This
is followed by a summary of some present and potential applications of immobilized enzymes.
Enzyme Immobilization

Many methods are available for enzyme immobilization. As we shall see below, the
immobilization method used greatly influences the properties of the resulting biocatalyst. Thus, the
selection of an immobilization strategy derives from process specifications for the catalyst
including such parameters as overall catalytic activity, effectiveness of catalyst utilization
deactivation and regeneration characteristics and, of course, cost. Also, toxicity of immobilization
reagents should be considered in connection with immobilization process waste disposal and
intended application of the immobilized enzyme catalyst.

Figure: Schematic illustration of several techniques for enzymes immobilization. (a) Chemical
methods, and (b) physical methods.

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Table: Interactions and carriers used for enzyme immobilization by adsorption

Interaction Adsorbents
Physical adsorption Activated carbon, silica gel, alumina, starch, clay, glass
Modified materials
Tannin-aminohexyl cellulose, Concanavalin A-Sepharose
Ionic binding Cation exchangers
CM – cellulose, Aberlite, CG – 50, Dowex 50
Anion exchangers
DEAE – cellulose, DEAE-Sephadex,
Polyaminopolystyrene, Amberlite IR - 45

CM = carboxymethyl; DEAE = diethylaminoethyl.

The various methods devised for enzyme immobilization may be subdivided into two
general classes: chemical methods, where covalent bonds are formed with enzyme, and physical
methods, where weaker interactions or containment of the enzymes are involved (figure).
Enzymes may be adsorbed on a variety of carriers (table), offering in some cases the practical
convenience of simple regeneration by removal of deactivated enzyme and reloading with fresh
active catalyst. If a support or entrapping material is used, its properties combined with those of
the enzyme and the immobilization procedure dictate overall catalyst properties.

Selecting a support for chemical or adsorption immobilization of enzyme depends first upon
its surface properties: Will the enzyme adsorb on the surface? Does the material possess functional
groups which can be used for bonding to the enzyme? If the native surface is not ideal, can it be
chemically modified or coated to facilitate enzyme attachment? Table lists several materials which
have been employed for covalent enzyme immobilization and some of their interesting surface
functional groups. Other materials which have been used as immobilized enzyme supports include
ceramics, glass and other metal oxides.

Protocols for covalent enzyme immobilization often begin with a surface modification or
activation step. Silanization, coating the surface with organic functional groups using an
organofunctional silane reagent (for example, (CH3CH2O, Si(CH2)3 where R is frequently – NH2), is a
widely used strategy for initial surface modification of inorganic supports. Such coatings for native
surface amino groups can be derivatized to aldehyde groups using glutaraldehyde, to arylamine
groups using p-nitrobenzoylchloride, or to carboxyl groups using succinic anhydride. Another
commonly studied surface modification is attachment to flexible spacer arm moieties (for example,
n-propyl amine) to the support, to which enzyme is subsequently linked. This may render the
surface more “flexible” so it can conform to the enzyme’s is subsequently linked. This may render
the surface a greater extend and potentially stabilizing native catalytic activity. Such modifications
may also be applied to alter the hydrophobicity or hydrophobicity of the support surface.

Table: Insoluble materials and some of their surface functional groups useful for covalent
enzyme attachment

Natural supports Synthetic supports

Cellulose (-OH) Polyacrylamide derivatives
CM-cellulose (-COOH) (Bio-Gel, Enzacryl) (-aromatic amino)
Agarose (Sepharose) (-OH) Polyaminopolystyrene (-NH2)
Dextran (Sephadex) (-OH) Maleic anhydride copolymers

Typical examples of immobilization chemistries which utilize these surface functionalities

and those appearing in table are summarized in figure. We should note that each step requires
suitably adjusted reaction conditions including pH, ionic strength, and reagent concentration. This
information is available in the chapter references along with extensive tabulations of specific
enzymes which have been immobilized by different methods on various supports. Which functional
groups on the protein are involved in covalent linkages to the support surface obviously depends
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on the immobilization chemistry applied. Taking advantage of the available repertoire of surface
modification techniques, typically there are several options for immobilizing a particular enzyme to
a particular support. Clearly, attachment to residues near or in the enzyme active site is to be
avoided.

When considering selection of a support and adjustment of its surface properties, one must
also consider interaction between the support surface and the reaction mixture. This interaction
may cause the fluid environment adjacent to the surface in contact with the enzyme to differ
substantially from the bulk fluid environment surrounding the immobilized enzyme catalyst. For
example, a charged support will increase local concentrations of oppositely charged ions. As a
result, the relationship between bulk solution pH and observed catalytic activity can be shifted
substantially relative to the pH activity function observed in solution. Similarly, the hydrophobicity
or hydrophilicity of the support will influence the local concentrations of solutes and solvents
according to their hydrophobicity/hydrophilicity.

Another important role of the support surface is indefining, directly and indirectly, the
molecular environment of the enzyme. That is, to some degree the enzyme will contact, at the
molecular level, the support surface. The remainder of the immobilized enzyme molecule will be in
contact with the local fluid environment which is influenced by the support as just outlined. The
enzyme molecule is structured so as to assume the proper configuration and corresponding activity
in its native biological environment. Clearly, it is possible by selection of the support for an
immobilized enzyme to attempt to mimic or to alter substantially the local enzyme environment
and thereby also to influence the catalytic activity of the enzyme and the effect of any modulating
factors on activity (and selectivity and stability). Efforts to understand enzyme-environment
interactions at the molecular level and to apply them for improved understanding of enzymes in
nature and for optimizing process biocatalysts are active topics of current research and
development.

2. Describe the Michaelis - Menten Kinetics

Now let us suppose that armed with a good set of experimental rate data we face the task of
representing it mathematically. From information of the type shown in Figure the
following qualitative features often emerge:

1. The rate of reaction is first order in concentration of substrate at relatively low

values of concentration. [Recall that if v = (const)(s n ) , n is the order of the reaction
rate].
2. As the substrate concentration is continually increased, the reaction order in
substrate diminishes continuously from one to zero.
3. The rate of reaction is proportional to the total amount of enzyme present.

Henri observed this behavior, and in 1902 propsed the rate equation
v s
v  max where v max  e 0 (3.3)
Km  s

Which exhibits all three features listed above. Notice that v  vmax / 2 when s is equal to
Km . To avoid confusion of the type found in some of the literature, we should strongly
emphasize that s is the concentration of free substrate in the reaction mixure, while e0 is
the concentration of the total amount of enzyme present in both the free and combined
forms (see below).

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Although Henri provided a theoretical explanation for Eq. using a hypothesize

pothesized reaction mechanism , his derivation and the similar one offered in 1913 by
Michaelis and Menten are now recognized as not rigorous in general. However, the general
methodology of the Michaelis-Menten treatment will find repeated useful (although still
not generally rigorously justified) application in derivation of more complicated kinetic
models later in this chapter. Consequently, we shall provide a brief summary of their
development before proceeding to others.

As a starting point, it is assumed that the enzyme E and substrate S combine to form
a complex ES, which then dissociates into product P and free (or uncombined) enzyme E:

SE
k1
k 1
ES (3.4a)
ES 
k2
 P+E (3.4b)

This mechanism includes the intermediate complex discussed above, as well as

regeneration of catalyst in this original form upon completion of the reaction sequence.
While perhaps considerable oversimplified, Eqs. are certainly reasonable.

Henri and Michaelis and Menten assumed that reaction is in equilibrium which, in
conjunction with the mass-action law for the kinetics of

(a) Figure: Kinetic data for enzyme-catalyzed reactions. (a) Enzyme concentration is
held constant when studying substrate concentration dependence; (b) the converse
holds for investigation on the influence of enzyme concentration

Molecular events, gives

se k
 1  K m  dissociation constant
 es  k1
Here, s,e, and (es) denote the concentration of S,E and ES, respectively. Decomposition of
the complex to product and free enzyme is assumed irreversible:
dp
v  k 2 (es)
dt
Since all enzyme present is either free or complexed, we also have

e + (es) = e0
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Where e0 is the total concentration of enzyme in the system. This is known from the
amount of enzyme initially charge into the reactor Equation with vmax equal to k2 e0 can
now be obtained by eliminating (es) and e from the three previous equations. We should
note here that a reaction described by Eq. is commonly referred to as having Michaelis-
Menten kinetics, although certainly other investigators made equal contributions to the
development add justification of this kinetic form. The parameter vmax is called the
maximum or limiting velocity, and Km is known as the Michaelis constant. While the
Michaelis-Menten equation successfully describes the kinetics of many enzyme-catalyzed
reactions, it is not universally valid. We shall explore the extensions and modifications
necessary for certain enzymes and reaction conditions in later sections and in the
problems.

Briggs and Haldane have provided the derivation of Eq. which later kinetic studies
and mathematical analyses have shown to be the most general. For reaction in a well-
mixed closed vessel we can write the following mass balances for substrate and the ES
ds
complex: v  k1se  k 1 (es)
dt

d(es)
and  k1se  (k 1  k 2 )(es)
dt

Using Eq. in the previous two equations gives a closed set: two simultaneous ordinary
differential equation in two unknowns, s and (es). The appropriate initial conditions are, of
course,
s(0)  S0 (es)(0) = 0

These equation cannot be solved analytically but they can be readily integrated on a
computer to find the concentrations of S, E, ES, and P as functions of time. In calculated
results illustrated in figure, it is evident that to a good approximation.

d(es)
0
dt

ES
k +1
k -1

ES 
k +2
PE

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with k+1  k+2

After a brief start-u period. In the key step in their analysis, Briggs and Haldane assumed
condition to be true. This assumption, commonly called the quasi-steady-state
approximation, can be proved valid for the present case of enzyme-catalyzed reaction in a
closed system provided that e0 / s0 is sufficiently small. If the initial substrate concentration
is not large compared with the total enzyme concentration, the assumption may break
down. In many instances, however, the amount of catalyst present is considerably less than
the amount of reactant, so that is an excellent approximation after start-up.

Proceeding from Equation as Briggs and Haldane did, it is possible to use Equation to
eliminate e and from the problem, leaving.

ds k2e0s
v 
dt [(k 1  k 2 ) / k1 ]  s

Consequently, the Michaelis-Menten form results, where Equation.

vmax  k2e0
k 1  k 2
and Km 
k1

Notice that Km no longer can be interpreted physically as a dissociation constant.

With the Michaelis-Menten rate expression at hand, the time course of the reaction
can now be determined analytically by integrating

ds v max s
 with s(0) = s0
dt K m  s

Figure:
Computed time course of batch hydrolysis of acetyl L-phenylalanine ether by
chymotrypsin. Considerable discrepancies between the exact solution and the quasi-
steady-state solution arise when e0 / s0 (=) is not sufficiently small.

to obtain
s0
v max t = s 0  s  K m ln
s

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Naturally this equation is most easily exploited indirectly by computing the reaction time t
required to reach various substrate concentrations, rather than vice versa.

It is instructive to compare the behavior predicted by Equation. With that obtained

without the quasi-steady-state approximation. As figure reveals, the deviation is significant
when the total enzyme concentration approaches s0 . Conseq2uently the Michaelis-Menten
equation should to be used in such cases. As another example, we show the dependence of
esterase activity on enzyme concentration in Figure. The linear dependence predicated by
the Michaelis-Menten model if followed initially but does not hold for large enzyme
concentrations. Remember that the slope at the linear portion is equal to k2s /(Km  s).

We should consider whether or not the quasi-steady-state approximation can be

justified in some cases of large e0 / s0 , perhaps with different rate constants. This could be
done if the ES complex tended to disappear much faster than it was formed, i.e., if Km were
very large relative to s0 Since the Michaelis constant is quite small, with typical values in
the range 102 to 10-5 , this case does not arise in may situations.

Consequently, if the enzyme concentration is comparable to s0 , we usually have no

proper justification for simplifying the kinetic model with the quasi-steady-state
approximation. Situations with relatively large enzyme concentration can occur in several
instances. If an enzyme reactor is operated under conditions where most of the substrate
is converted, a falls to a value comparable to e0 and the Michaelis-Menten equation may be
inappropriate for the final stages of reaction.

Figure: These data on esterase activity show deviations from Michaelis-Menten

kinetics at large values of initial enzyme content.

Fortunately, the reaction has become slow here, and this situation is consequently
seldom of interest. Also in the case of enzyme reactions at interfaces, which we shall
investigate in Sec., the substrate concentration in the neighborhood of the enzyme may be
quite small. Here, the rate of substrate transport to the interface must also be considered.
Possible pitfalls in the Michaelis-Menten model have come to light only very recently, and
consequently it is still commonly employed for analyzing and designing enzyme reactors.
Although they lack rigorous justification in some situations, the equilibrium assumption
and the Michaelis-Menten rate equation have proved to be valuable working tools. We
shall often employ them in this spirit.

While on the subject of simple Michaelis-Menten kinetics, we should emphasize

that exactly the same mathematical form is widely used for expressing the rates of many
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solid-catalyzed reactions. In chemical engineering practice, kinetics described by Equation

is called Langmuir-Hinshelwood or Hougen-Watson kinetics. Since reactions involving
synthetic solid catalysts are widespread throughout the chemical and petroleum industries,
a large effort has been devoted to the design and analysis of catalytic ractors. Much of this
work has employed Langmuir-Hinshelwood kinetics and is therefore directly applicatble to
enzyme-catalyzed reactions described by Michaelis-Menten kinetics. In the remainder of
our studies, we shall continue to observe analogies, similarities, and identities between
classical chemical engineering and biochemical technology. Also, of course, we shall
encounter many novel features of biological processes.

3. Explain Enzymes – Substrate Complex (or) mechanism of enzymatic reactions?

THE ENZYME-SUBSTRATE COMPLEX AND ENZYEM ACTING

There is no single theory currently available which accounts for the unusual
specificity and activity of enzyme catalysis. However, there are a number of plausible idas
supported by experimental evidence for a few specific enzymes. Probably, then, all or some
collection of these phenomena actin together combine to give enzymes their special
properties. In this section we shall outline some of these concepts. Our review will
necessarily be brief, and the interested reader should consult the references for further
details. Since all the theories mentioned here are at best partial successes, we must be
wary of attempting to synthesize a single theory of enzyme activity.

Verified numerous experimental investigations involving such diverse techniques as

x-ray crystallography, spectroscopy, and electron-spin resonance is that existence of a
substrate-enzyme complex. The substrate binds to a specific region of the enzyme called
the active site, where rectin occurs ad products are released; binding to create the complex
is sometimes due to the type of weak attractive force outlined in Sec. 2.4.3 Although
covalent attachments are known for some cases. As shown schematically in Figures , the
complex is figure are the hydrogen bonds which form between the substrate and groups
widely separated in the amino acid chain of the enzyme.

This example also nicely illustrates the notion of an active site. The protein
molecule is folded in such a way that a group of reactive amino acid side chains in the
enzyme presents a very specific site to the substrate. The reactive groups encountered in
enzymes include the R group of Asp, Cys, Glu, His, Lys, Met, Ser, Thr, and the end amino and
carboxyl functions. Since the number of such groups near the substrate is typically 20 (far
less than the total number of amino acid residuce present), only a small fraction of the
enzyme is believed to participate directly in the enzyme’s active site. Large enzymes may
have more than one active site. Many of the remaining amino acids determine the folding
along a chain of amino acids (secondary structure) and the placement of one part of a
folded chain next to another (tertiary structure), which help create the active site itself.

While some of the ideas described below are still somewhat controversial, we
should emphasize that the notions of active sites and the enzyme-substrate complex are
universally accepted and form the starting point for most theories of enzyme action. These
concepts will also be the cornerstons of our analysis of enzyme kinetics.

Two different aspects of current thinking on enzyme activity are shown

schematically in figure. Enzymes can hold substrates so that their reactive regions are
close to each other and to the enzyme’s catalytic groups. This feature, which quite logically
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can accelerate a chemical reaction, is known as the proximity effect. Consider now that the
two substrates are not spherically symmetrical molecules. Consequently, reaction will
occur only when the molecules come together at the proper orientation so that the reactive
atoms or groups are in close juxtaposition.

Figure:
A view of the active site of lysozyme, showing a hexasccharide substrate in heavy
lines. Larger and smaller circles denote oxygen and nitrogen atoms, respectively,
and hydrogen bonds are indicated by dashed lines.

Figure:
An enzyme may accelerate reaction by holding two substrates close to each othe
r(proximity effect) and at an advantageous angle (orientation effect).

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Enzymes are believed to bind substrates in especially favorable positions, thereby

contribution an orientation effect, which accelerates the rate of reaction. Also called orbital
steering, this phenomenon has qualitative merit as a contributing factor to enzyme
catalysis. The quantitative magnitude of its effect, however, is still difficult to assess in
general.

Before a brief foray into the chemistry of enzyme action, we should mention one
other hypothesis related to enzyme geometry. It is known that for some enzymes the
binding of substrate causes the shape of the enzyme to change slightly. Studies of the
three-dimensional structure of the enzymes lysozyme and canboxpeptidase A with an
without substrates have shown a change in enzyme conformation upon addition of the
substrate. This induced fit of enzyme and substrate may add to the catalytic process. There
are also more sophisticated extension of the induced-fit model, in which a number of
different intermediate enzyme-substrate complexes are formed as the reaction progresses.
A slightly elastic and flexible enzyme molecule would have the ability to make delicate
adjustments in the position of its catalytic groups to hasten the transformation of each
intermediate. Since direct measurements of the influence of substrate binding on enzyme
structure are available only for a few enzymes, it is possible that a substrate-induced
change in active-site configuration may be a general characteristic of enzyme catalysis.

Catalytic processes well known to the organic chemist also appear to be at work in
some enzymes. One of these is general acid-base catalysis, where the catalyst accepts or
donates protons somewhere in the overall in overall catalytic process. In one of the few
enzymes for which a reasonable complete catalytic sequence is proposed, this mode of
catalysis is present. Chymotrypsin, derived from the pancreas, is a proteolytic (protein-
hydrolyzing) enzyme with specificity for peptide bonds where the carbonyl side is a
tyrosine, or phenylalanine residue. Water is believed to serve as a proton-transfer agent in
both the general acid- and general base-catalyzed portions of the chymotrypsin
mechanism. Several reactions important to cellular chemistry, including carbonyl addition
and ester hydrolysis, are in principle amenable to general acid-base catalysis.

Participating in enzyme catalysis may be a number of other phenomena such as

covalent catalysis, electrostatic catalysis, multifunctional catalysis, and solvent effect (recall
the oil-drop structure from Chap. 2). Details on these mechanisms available in the
references reveal that, like the notions reviewed here, they are probably involved in some
enzyme-catalyzed reactions. From the possible involvement of these factors and others, it
is not surprising that no one has yet devised a simple general scheme for assessing their
combined influence and relative importance. Fortunately, armed only with the basic idea
of an enzyme substrate complex as an essential reaction intermediate, we shall be able to
formulate useful rate expressions for enzyme-catalyzed reactions.

4. Describe the Modulation and Regulation of Enzymatic Activity?

Chemical species other than the substrate can combine with enzymes to alter or modulate
their catalytic activity. Such substances, called modulators or effectors, may be normal constituents
of the cell. In other cases they enter form the cell’s environment or act on isolated enzymes.
Although most of our attention in this section will be concentra6ted on inhibition, where the
modulator decreases activity, cases of enzyme activation by effectors are also known.
The combination on and enzyme with an effector is itself a chemical reaction and may
therefore be fully reversible, partially reversible, or essentially irreversible. Known examples of
irreversible inhibitors include poisons such as cyanide ions, which deactivate xanthine oxidase, and
a group of chemicals collectively termed nerve gases, which deactivate cholinesterases (enzymes
which are an intetermed nerve transmission and thus of motor ability). If the inhibitor acts
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irreversibly, the Michaelis-Menten approach to inhibitor-influenced kinetics cannot be used since

this method assumes equilibrium between the free and complexed forms. Often, irreversible
inhibition increases with time as more and more enzyme molecules are gradually deactivated.
Other cases, more difficult to detect, involve the partial deactivation of enzyme. In such an instance,
the inhibited enzyme retains catalytic activity although at a level reduced from the pure form.
Section mentioned the remarkable chemical capabilities of most cicroor4ganisms which,
supplied with only a few relatively simple precursors, manufacture a vast array of complex
molecules. In order to perform this feat, it is necessary for the supply of precursors to be
proportioned very efficiently among the many synthesis routes leading to many end products.
Optimum utilization of the available chemical raw materials in most instances requires that only
the necessary amount of any end product be manufactured. If enough of one monomer is present,
for example, its synthesis should be curtailed and attention devoted to making other compounds
which are in relatively short supply.
Reversible modulation of enzyme activity is one control mechanism employed by the cell
to achieve efficient use of nutrients. (The other major control device is discussed in sec.) The most
intriguing examples of enzyme regulation involve interconnected networks of reactions with
several control loops, but their analysis must wait until we have studied the basic chemistry of
cellular operation in Chap.5. For present purposes, we shall employ control of a single sequence of
reactions as an example. Figure shows a five-step sequence for the biosynthesis of the amino acid
L-isoleucine. Regulation of this sequence is achieved by feedback inhibition: the final product, L-
isoleucine, inhibits the activity of the first enzyme in the path. Thus, if the final product begins to
build up, the biosynthesis process will be stopped.
Since the reactions catalyzed by enzymes E 2 through E 5 are essentially at equilibrium and
the first reaction is “irreversible” under cellular conditions, the response of this control device is
especially fast. Indeed, it is a general property of most regulatory enzymes that they catalyze
“irreversible” reactions. Also, it should be obvious from the biological context that such “natural”
enzyme modulation must be reversible. For example, if L-isoleucine is depleted by its use in
protein synthesis, inhibition of enzyme E1 must be quickly relaxed so that the required supply of
the amino acid is restored.

Figure:
In this feedback-inhibition system, the activity of enzyme E1 (L-threonine deaminase) is
reduced b the presence of the end product, L-isoleucine. (
P1    ketobutyrate, P2    acetohydroxybutyrate, P3   , -dihydroxy--methylvalerate,

P4    keto   methylvalerate )

Table Partial classification of reversible inhibitors

Type Description Result

Ia Fully competitive Inhibitor adsorbs at Increase in apparent
substrate binding site value of Km
B Partially competitive Inhibitor and substrate Increase in apparent
combine with different value of Km
groups; inhibitor
binding affects
substrate binding

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IIa Noncompetitive Inhibitor binding does No change in Km ,

not affect ES affinity, decrease of v max
but ternary EIS
(enzyme-inhibitor-
substrate) complex
does not decompose
into products.
B Noncompetitive Same as IIa except than No change in Km ,
EIS decomposes into decrease of v max
product at a finite rate
different from that of
ES
III Mixed inhibitor Affects both Km and
vmax

In the case or reversible inhibition, the approaches described in Sec. prove quite fruitful.
Since many isolated enzyme-substrate systems exhibit Michaelis-Menten kinetics, it is traditional to
classify inhibitors by their influence on the Michaelis-Menten equation parameters v max and K m .

Reversible inhibitors are termed competitive if their presence increases the value of Km .
But does not alter v max . The effect of such inhibitors can be countered or reversed by increasing
the substrate concentration. On the other hand, by rendering the enzyme or the enzyme-substrate
complex inactive, a noncompetitive inhibitor decreases the v max of the enzyme but does not alter
the Km value. Consequently increasing the substrate concentration to any level cannot produce as
great a reaction rate as possible with the uninhibited enzyme. Common noncompetitive inhibitors
are heavy-metal ions, which combine reversibly with the sulfhydryl (-SH) group of cysteine
residues.

Several different combinations and variations on the two basic types of reversible
inhibitors are known. Some of these are listed in Table. Experimental discrimination between these
possibilities will be discusses shortly. First, however, we shall briefly review some of the current
theories and available data on the mechanisms of modulator action.

St. Joseph’s College of Engineering 14 Department Of Chemical Engineering