ENZYMES

ENZYMES
Most Enzymes Are Proteins
 With the exception of a small group of catalytic RNA molecules , all enzymes are proteins.
 Some enzymes require no chemical groups for activity other than their amino acid residues. Others require an additional chemical component called a cofactor—either one or more inorganic ions, such as Fe2+, Mg2+, Mn2+, or Zn2_+(Table 6–1), or a complex organic or metalloorganic molecule called a coenzyme Some enzymes require both a coenzyme and one or more metal ions for activity.
 A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group.
 A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme.
 The protein part of such an enzyme is called the apoenzyme or apoprotein.




Enzymes Are Classified by the Reactions They Catalyze




Chemical Nature of Enzyme
Substance Hydrolyzed And The Group Involved.
1. Carbohydrate-hydrolyzing enzymes
(a) Glycosidases—cellulase, amylase, sucrase, lactase, maltase
(b) -glucorinidase
2. Protein-hydrolyzing enzymes
(a) Peptide bonds
I. Endopeptidases
 Animals— pepsin, trypsin, rennin
 Plants—papain ficin, bromolin
II. Exopeptidases—dipeptidase, tripeptidase
(b) Nonpeptide C—N linkages (amidases) urease, arginase, glutaminase
3. Lipid-hyrolyzing enzymes
 lipases, esterases, lecithinases
4. Other ester-hydrolyzing enzymes
(a) Phosphatases
(b) Cholinesterases
(c) Chlorophyllases
(d) Sulfatases
(e) Pectinesterases
(f) Methylases
5. Oxidation-reduction enzymes
 hydrases, mutases, oxidases, dehydrogenases, peroxidases
6. Miscellaneous enzymes
 catalase, carboxylase, carbonic anhydrase, thiaminase, transpeptidase























































Chemical composition of the enzyme.
 Based on their chemical composition, the enzymes have been classified into following three categories :
1. Enzyme molecule consisting of protein only— e.g., pepsin, trypsin, urease, papain, amylase etc.
2. Enzyme molecule containing a protein and a cation— e.g., carbonic anhydrase (containing Zn2+ as cation), arginase (Mn2+), tyrosinase (Cu2+) etc.
3. Enzyme molecule containing a protein and a nonprotein organic compound known as prosthetic group—Tauber (1950) has further subdivided them, on the basis of the nature of prosthetic group involved :

(a) Iron prophyrin enzymes— catalase, cytochrome c peroxidase I and II.
(b) Flavoprotein enzymes— glycine oxidase, pyruvate oxidase, histamine.
(c) Diphosphothiamin enzymes — -carboxylase, pyruvate mutase.
(d) Enzymes requiring other coenzymes— phosphorylase, amino acid decarboxylase.

How Enzymes Work
The difference between the energy levels of the ground state and the transition state is the activation energy, ∆G◦.
The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction.





















Catalysts enhance reaction rates by lowering activation energies. Enzymes are no exception to the rule that catalysts do not affect reaction equilibria.
The role of enzymes is to accelerate the interconversion of S and P. The enzyme is not used up in the process, and the equilibrium point is unaffected.
When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation energy; this is called the rate-limiting step.

A Few Principles Explain the Catalytic Power and Specificity of Enzymes
o Enzymes are extraordinary catalysts how can these enormous and highly selective rate enhancements be explained? What is the source of the energy for the dramatic lowering of the activation energies for specific reactions?

 The answer to these questions has two distinct but interwoven parts.
1. The first lies in the rearrangements of covalent bonds during an enzyme-catalyzed reaction.Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and activate it for reaction, or a group may be transiently transferred from the substrate to the enzyme.



Covalent interactions between enzymes and substrates lower the activation energy (and thereby accelerate the reaction) by providing an alternative, lower-energy reaction path.




2. The second part of the explanation lies in the noncovalent interactions between enzyme and substrate.
Much of the energy required to lower activation energies is derived from weak, noncovalent interactions between substrate and enzyme.
What really sets enzymes apart from most other catalysts is the formation of a
specific ES complex.
The interaction between substrate and enzyme in this complex is mediated by the same forces that stabilize protein structure, including hydrogen bonds and hydrophobic and ionic interactions.

Formation of each weak interaction in the ES complex is accompanied by release of a small amount of free energy that provides a degree of stability to the interaction.
The energy derived from enzyme-substrate interaction is called binding energy, ∆GB. Its significance extends beyond a simple stabilization of the enzyme-substrate interaction. Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions.

o Two fundamental and interrelated principles provide a general explanation for how enzymes use noncovalent binding energy

Much of the catalytic power of enzymes is ultimately derived from the free energy released in forming many weak bonds and interactions between an enzyme and its substrate. This binding energy contributes to specificity as well as to catalysis.
Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per se but to the transition states through which substrates pass as they are converted to products during an enzymatic reaction.
Consider what needs to occur for a reaction to take place.
weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis. formation of weak bonds between substrate and enzyme also results in desolvation of the substrate.
Enzyme-substrate interactions replace most or all of the hydrogen bonds between the substrate and water.
A large restriction in the relative motions of two substrates that are to react, or entropy reduction, is one obvious benefit of binding them to an enzyme.

The enzyme itself usually undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate. This is referred to as induced fit.
Specific Catalytic Groups Contribute to Catalysis General Acid-Base Catalysis
Charged intermediates can often be stabilized by the transfer of protons to or from the substrate or intermediate to form a species that breaks down more readily to products.
For nonenzymatic reactions, the proton transfers can involve either the constituents of water alone or other weak proton donors or acceptors.
Catalysis of this type that uses only the H+ (H3O+) or OH- ions present in water is referred to as specific acid-base catalysis. If protons are transferred between the intermediate and water faster than the intermediate breaks down to reactants, the intermediate is effectively stabilized every time it forms. No additional catalysis mediated by other proton acceptors or donors will occur.
In many cases, however, water is not enough. The term general acid-base catalysis refers to proton transfers mediated by other classes of molecules. For nonenzymatic reactions in aqueous solutions, this occurs only when the unstable reaction intermediate breaks down to reactants faster than protons can be transferred to or from water.
Many weak organic acids can supplement water as proton donors in this situation, or weak organic bases can serve as proton acceptors.
The presence of alternative proton donors (HA) or acceptors (B ) increases the rate of the reaction.




Covalent Catalysis-- In covalent catalysis, a transient covalent bond is formed between the enzyme and the substrate. Consider the hydrolysis of a bond between
groups A and B:

In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes


This alters the pathway of the reaction, and it results in catalysis only when the new pathway has a lower activation energy than the uncatalyzed pathway.

Metal Ion Catalysis

Enzyme Kinetics:
Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions [S]
One simplifying approach in kinetics experiments is to measure the initial rate (or initial velocity), designated V0, when [S] is much greater than the concentration of enzyme, [E].
At relatively low concentrations of substrate, V0 increases almost linearly with an increase in [S]. At higher substrate concentrations, V0 increases by smaller and smaller amounts in response to increases in [S].


FIGURE 6–11( rectangular hyperbola) Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction. Vmax is extrapolated from the plot, because V0 approaches but never quite reaches Vmax. The substrate concentration at which V0 is half maximal is Km, the Michaelis constant. The concentration of enzyme in an experiment such as this is generally so low that [S] >>> [E] even when [S] is described as low or relatively low. The units shown are typical for enzyme-catalyzed reactions and are given only to help illustrate the meaning of V0 and [S]. (Note that the curve describes part of a rectangular hyperbola, with one asymptote at Vmax. If the curve were continued below [S] = 0, it would approach a vertical asymptote at [S] = -Km.)


The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed Quantitatively


V0 is determined by the breakdown of ES to form product, which is determined by [ES]:


[Et] =representing the total enzyme concentration (the sum of free and substrate-bound enzyme).
[Et] - [ES]= Free or unbound enzyme

Step 1 The rates of formation and breakdown of ES are determined by the steps governed by the rate constants k1 (formation) and k_1 + k2 (breakdown), according to the expressions.

Rate of ES formation = k1([Et] - [ES])[S]

Rate of ES breakdown = k_1[ES] + k2[ES]

Step 2 We now make an important assumption: that the initial rate of reaction reflects a steady state in which [ES] is constant—that is, the rate of formation of ES is equal to the rate of its breakdown. This is called the steady-state assumption.

k1([Et] -[ES])[S] = k_1[ES] + k2[ES]






The term (k2 + k_1)/k1 is defined as the Michaelis constant, Km. Substituting this into Equation 6–18 simplifies the expression to


(

This equation can be further simplified. Because the maximum velocity occurs when the enzyme is saturated (that is, with [ES] = [Et]) Vmax can be defined as k2[Et].


Note that Km has units of concentration.


An important numerical relationship emerges from the Michaelis-Menten equation in the special case when V0 is exactly one-half Vmax (Fig. 6–12). Then






This form of the Michaelis-Menten equation is called the Lineweaver-Burk equation.




The most important exceptions to Michaelis-Menten kinetics are the regulatory enzymes.


BISUBSTRATE REACTIONS

 Almost all of these so-called bisubstrate reactions are either transferase reactions in which the enzyme catalyzes the transfer of a specific functional group, X, from one of the substrates to the other :




or oxidation-reduction reactions in which reducing equivalents are transferred between the two substrates.
For example, in the peptide hydrolysis reaction catalyzed by trypsin, the peptide carbonyl group with its pendent polypeptide chain is transferred from the peptide nitrogen atom to a water molecule :



Similarly, in the alcohol dehydrogenase reaction, a hydride ion is formally transferred from ethanol to NAD+ :




X

In such reactions, the sequence of binding of the substrate to the enzyme molecule may be of different
types. Depending on the sequence of binding of the substrate to the enzyme, W.W. Cleland (1967)
propounded 4 types of mechanisms and explained them with the help of what are commonly called ‘Cleland
diagrams’ (Fig. 18−22). Cleland’s nomenclatural concept is based on the following 5 conventions :
1. The substrates are designated by the letters A, B, C and D in the order that they add to the enzyme.
2. The products are denoted by the letters P, Q, R and S in the order that they leave the enzyme.
3. The enzyme is indicated by the letter E. The intermediary stable forms of enzymes, that are produced and then disappear during the course of reaction, are represented by the letters F, G, H etc.
4.The enzyme-substrate complexes are shown in parenthesis.



Types of Bi Bi reactions
In this section, we shall deal with reactions that require two substrates and yield two products,
that is Bi Bi reactions. It may, however , be remembered that there are numerous examples of even
more complex reactions. Enzyme-catalyzed group-transfer Bi Bi reactions fall under two major
mechanistic classifications.
1. Sequential reactions
Reactions in which all substrates combine with the enzyme before a reaction can occur and
products be released are known as sequential reactions. In such reactions, the group being transferred,
X, is directly passed from A (= P-X) to B, yielding P and Q ( = B -X). Hence, such reactions are also
called single-displacement reactions.
Sequential reactions can be further classified into 2 types :
(a) those with a compulsory order of substrate addition to the enzyme, which are said to have an
ordered mechanism, and
(b) those with no preference for the order of substrate addition, which are described as having a
random mechanism.
In the ordered mechanism, the binding of the first substrate is apparently required for the enzyme to form the binding site for the second substrate, whereas for the random mechanism, both binding
sites are present on the free enzyme.
2. Ping pong reactions
Reactions in which one or more products are released before all substrates have been added are
known as ping pong reactions. In such a reaction, a functional group X of the first substrate
A (= PX) is displaced from the substrate E to yield the first product P and a stable enzyme form
F (= E------X) in which X is tightly (often covalently) bound to the enzyme (ping). In the second stage
of the reaction, X is displaced from the enzyme by the second substrate B to yield the second product
Q (= BX), thereby regenerating the original form of the enzyme, E (pong). Such reactions are,
hence, also known as double-displacement reactions. A notable feature of ping pong Bi Bi reactions
is that the substrates A and B do not encounter one another on the surface of the enzyme.
The salient features of the mechanisms underlying these different reaction types are described
hereunder :
1. The ordered sequential mechanism. This mechanism envisages that the first substrate (A)
binds with the enzyme (E), followed by the second substrate (B). The substrate A which combines
first is called the leading substrate and the substrate B which combines later is termed the following
substrate. A ternary complex (EAB) between enzyme and the two substrates is formed. In this complex,
both substrates are converted to products (P and Q) and then liberated in the same sequence their
corresponding substrate had combined, i.e., the product P is released first followed by Q. Many
NAD+- and NADP+-requiring dehydrogenases follow an ordered Bi Bi mechanism in which the
coenzyme is the leading substrate.
2. The random sequential mechanism. In this mechanism, there is no definite sequence of
substrate association to the enzyme. They can bind in any order and the products from the ternary
complex can also be released in any order. Some dehydrogenases and kinases operate through this
mechanism.
3. Theorell−Chance mechanism. It is a variant of the ordered sequential mechanism in which
the ternary complex (EAB) is not at all formed. This mechanism operates in the reaction catalyzed by
alcohol dehydrogenase (E.C. No. 1.1.1.1).
4. The ping pong mechanism. This mechanism envisages that one substrate binds and one
product is released before the second substrate can bind to the enzyme and may release the second
substrate. Thus, here the enzyme acts as a board and the substrates act as ping pong balls. Many
enzymes, including glutamate dehydrogenase (E.C. No. 1.4.1.2), chymotrypsin, transaminases and
some flavoenzymes obey ping pong mechanism.
C. Kinetics of Bi Bi Reactions
Steady state kinetic measurements can be utilized to distinguish among the foregoing bisubstrate
mechanisms. For doing so, one must first derive their rate equations. This can be done in much the
same way as for single-substrate-enzymes, i.e., solving a set of simultaneous linear equations consisting
of an equation expressing the steady state condition for each kinetically distinct enzyme complex plus
one equation representing the conservation condition for the enzyme. This, indeed, is a more complex
situation for bisubstrate enzymes than it is for single-substrate or monosubstrate enzymes.
The rate equations for the above-described bisubstrate mechanisms in the absence of products
are given below in double reciprocal form.
For ordered Bi Bi reactions :



For random Bi Bi reactions :
The rate equation for the general random Bi Bi reaction is quite complicated. However, in the
special case that both substrates are in rapid and independent equilibrium with the enzyme; that is,
the EAB–EPQ interconversion is rate determining, the initial rate equation reduces to the following
relatively simple form. This mechanism is known as the rapid equilibrium random Bi Bi mechanism :



For ping pong Bi Bi reactions :