Enzymes are central to every biochemical process. Acting in organized sequences, they catalyze the hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform chemical energy, and make biological macromolecules from simple precursors. The study of enzymes has immense practical importance. In some diseases, especially inheritable genetic disorders, there may be a deficiency or even a total absence of one or more enzymes. Other disease conditions may be caused by excessive activity of an enzyme.
Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosing certain illnesses. Many drugs act through interactions with enzymes. Enzymes are also important practical tools in chemical engineering, food technology, and agriculture.
Enzymes, the most remarkable and highly specialized proteins. Enzymes have extraordinary catalytic power, often far greater than that of synthetic or inorganic catalysts. They have a high degree of specificity for their substrates, they accelerate chemical reactions tremendously, and they function in aqueous solutions under very mild conditions of temperature and pH. Few nonbiological catalysts have all these properties.
Proteinaceous enzyme can be divided into two general classes 1st simple enzyme consist entirely of amino acid and 2nd conjugated enzyme consist of protein as well as non protein component. The non protein component is called a cofactor which is required for catalytic activity. Removal of cofactor from conjugated enzyme leaves only protein component, called apoenzyme, which is generally biologically inactive.
The complete biologically active conjugated enzyme ( simple enzyme plus cofactor) is called holoenzyme. A cofactor can be linked to protein portion of the enzyme either covalently or non covalently. Some cofactors are simple metal ions and other cofactor are complex organic compound which are also called coenzymes. Many coenzymes are vitamins or contain vitamins as a part of their structure. Some coenzymes are only transiently associated with a given enzymes so that they can function as cosubstrates.
Coenzymes which are tightly associated with proteins covalently or non covalently are called prosthetic group.
CLASSIFICATION OF ENZYME
Many enzymes have common names that provide little information about the reactions that they catalyze. For example, a proteolytic enzyme secreted by the pancreas is called trypsin. Most other enzymes are named for their substrates and for the reactions that they catalyze, with the suffix “ase” added. Thus, an ATPase is an enzyme that breaks down ATP, whereas ATP synthase is an enzyme that synthesizes ATP. To bring some consistency to the classification of enzymes, in 1964 the International Union of Biochemistry established an Enzyme Commission to develop a nomenclature for enzymes.
Reactions were divided into six major groups numbered 1 to 6. These groups were subdivided and further subdivided, so that a four-digit number preceded by the letters EC for Enzyme Commission could precisely identify all enzymes.
ATP + NMP –> ADP + NDP
NMP kinase transfers a phosphoryl group from ATP to NMP to form a nucleoside diphosphate (NDP) and ADP. Consequently, it is a transferase, or member of group 2. Many groups in addition to phosphoryl groups, such as sugars and carbon units, can be transferred. Transferases that shift a phosphoryl group are designated 2.7. Various functional groups can accept the phosphoryl group. If a phosphate is the acceptor, the transferase is designated 2.7.4.
The final number designates the acceptor more precisely. In regard to NMP kinase, a nucleoside monophosphate is the acceptor, and the enzyme’s designation is EC 126.96.36.199. Although the common names are used routinely, the classification number is used when the precise identity of the enzyme might be ambiguous.
THE MICHAELIS-MENTEN MODEL ACCOUNTS FOR THE BASIC KINETIC PROPERTIES OF MANY ENZYMES
A key factor affecting the rate of a reaction catalyzed by an enzyme is the concentration of substrate, [S]. However, studying the effects of substrate concentration is complicated by the fact that [S] changes during the course of an in vitro reaction as substrate is converted to product. One simplifying approach in kinetics experiments is to measure the initial rate (or initial velocity), designated V0.
In a typical reaction, the enzyme may be present in nanomolar quantities, whereas [S] may be five or six orders of magnitude higher. If only the beginning of the reaction is monitored, over a period in which only a small percentage of the available substrate is converted to product, [S] can be regarded as constant, to a reasonable approximation. V0 can then be explored as a function of [S], which is adjusted by the investigator. .
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]. Finally, a point is reached beyond which increases in V0 are vanishingly small as [S] increases. This plateau-like V0 region is close to the maximum velocity, Vmax .
Leonor Michaelis and Maud Menten in 1913 postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step :-
The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P
Because the slower second reaction must limit the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step—that is, ES
The maximum initial rate of the catalyzed reaction (Vmax ) is observed when virtually all the enzyme is present as the ES complex and [E] is vanishingly small. Under these conditions, the enzyme is “saturated” with its substrate, so that further increases in [S] have no effect on rate.
This condition exists when [S] is sufficiently high that essentially all the free enzyme has been converted to the ES form. After the ES complex breaks down to yield the product P, the enzyme is free to catalyze the reaction of another molecule of substrate (and will do so rapidly under saturating conditions). The saturation effect is a distinguishing characteristic of enzymatic catalysts and is responsible for the plateau.
The curve expressing the relationship between [S] and V0 has the same general shape for most enzymes (it approaches a rectangular hyperbola), which can be expressed algebraically by the Michaelis-Menten equation. Michaelis and Menten derived this equation starting from their basic hypothesis that the rate-limiting step in enzymatic reactions is the breakdown of the ES complex to product and free enzyme. The equation is All these terms—[S], V0 , Vmax , and a constant, Km, called the Michaelis constant—are readily measured experimentally.
The overall reaction then reduces to
Because [ES] is not easily measured experimentally, we must begin by finding an alternative expression for this term. First, we introduce the term [Et ], representing the total enzyme concentration (the sum of free and substrate-bound enzyme). Free or unbound enzyme [E] can then be represented by [Et ] − [ES]. Also, because [S] is ordinarily far greater than [Et ], the amount of substrate bound by the enzyme at any given time is negligible compared with the total [S]. With these conditions in mind, the following steps lead us to an expression for V0 in terms of easily measurable parameters.
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 to reactants and products, respectively), according to the expressions
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.
First, the left side is multiplied out and the right side simplified to give
Adding the term k1 [ES][S] to both sides of the equation and simplifying gives
We then solve this equation for [ES]
The term (k−1 + k2 )/k1 is defined as the Michaelis constant, Km. Substituting this into Equation 6-18 simplifies the expression to
We can now express V0 in terms of [ES].
This equation can be further simplified. Because the maximum velocity occurs when the enzyme is saturated (that is, when [ES] = [Et ]), Vmax can be defined as k2 [Et ].
This is the Michaelis-Menten equation, the rate equation for a one substrate enzyme-catalyzed reaction. It is a statement of the quantitative relationship between the initial velocity V0 , the maximum velocity Vmax , and the initial substrate concentration [S], all related through the Michaelis constant Km.
An important numerical relationship emerges from the Michaelis-Menten equation in the special case when V0 is exactly one-half Vmax. Then
Solving for Km, we get Km + [S] = 2[S], or
This is a very useful, practical definition of Km: Km is equivalent to the substrate concentration at which V0 is one-half Vmax .
When the substrate concentration is much greater than KM, the rate of catalysis is equal to kcat However, most enzymes are not normally saturated with substrate. Under physiological conditions, the [S]/KM ratio is typically between 0.01 and 1.0. When [S]<< KM, the enzymatic rate is much less than kcat because most of the active sites are unoccupied.
when [S],<< KM, the concentration of free enzyme, [E], is nearly equal to the total concentration of enzyme
Thus, when [S] KM, the enzymatic velocity depends on the values of kcat/KM, [S], and [E]T. Under these conditions, kcat/KM is the rate constant for the interaction of S and E and can be used as a measure of catalytic efficiency. For instance, by using kcat/KM values, one can compare an enzyme’s preference for different substrates.
- Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 10 5 to 10 17
- Enzyme-catalyzed reactions are characterized by the formation of a complex between substrate and enzyme (an ES complex). Substrate binding occurs in a pocket on the enzyme called the active site.
- A significant part of the energy used for enzymatic rate enhancements is derived from weak interactions (hydrogen bonds, aggregation due to the hydrophobic effect, and ionic interactions) between substrate and enzyme. 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.
- Most enzymes have certain kinetic properties in common. When substrate is added to an enzyme, the reaction rapidly achieves a steady state in which the rate at which the ES complex forms balances the rate at which it breaks down. As [S] increases, the steady-state activity of a fixed concentration of enzyme increases in a hyperbolic fashion to approach a characteristic maximum rate, Vmax , at which essentially all the enzyme has formed a complex with substrate.
- The substrate concentration that results in a reaction rate equal to one-half Vmax is the Michaelis constant Km, which is characteristic for each enzyme acting on a given substrate. The Michaelis-Menten equation relates initial velocity to [S] and Vmax through the constant Km. MichaelisMenten kinetics is also called steady-state kinetics.
- Km and Vmax have different meanings for different enzymes. The limiting rate of an enzyme-catalyzed reaction at saturation is described by the constant kcat , the turnover number. The ratio kcat /Km provides a good measure of catalytic efficiency. The Michaelis-Menten equation is also applicable to bisubstrate reactions, which occur by ternary complex or Ping-Pong (double displacement) pathways.
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:- Article Written By Zahra Madraswala