Enzymes
- 6.1 An Introduction to Enzymes 183
- 6.2 How Enzymes Work 186
- 6.3 Enzyme Kinetics as an Approach to Understanding mechanism 194
- 64 Examples of Enzymatic Reactions 205
- 6.5 Regulatory Enzymes 220
Introduction:-
- There are two fundamental conditions for life.
- First, the organism must be able to self-replicate;
- second, it must be able to catalyze chemical reactions efficiently and selectively.
- The central importance of catalysis may seem surprising, but it is easy to demonstrate.
- As living systems make use of energy from the environment.
- Many of them, for example, consume substantial amounts of sucrose-common table sugar as a kind of fuel, usually in the form of sweetened foods and drinks.
- The conversion of sucrose to CO2, and H₂O in the presence of oxygen is a highly exergonic process, releasing free energy that we can use to think, move, taste and see.
- However, a bag of sugar can remain on the shelf for years without any obvious conversion to CO₂ and H2O.
- Although this chemical process is thermodynamically favorable, it is very slow!
- Yet when sucrose is consumed by a human (or almost any other organism), it releases its chemical energy in seconds.
- The difference is catalysis.
- Without catalysis, chemical reactions such as sucrose oxidation could not occur on a useful time scale, and thus could not sustain life.
- In this chapter, then we turn our attention to the reaction catalysts of biological systems: the enzymes, the most remarkable and highly specialized protein.
- 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.
- 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.
- We begin with descriptions of the properties of enzymes and the principles underlying their catalytic power, then introduce enzyme kineties, a discipline that provides much of the framework for any discussion of enzymes.
- Specific examples of enzyme mechanisms are then provided, illustrating principles introduced earlier in the chapter.
- We end with a discussion of how enzyme activity is regulated
6.1 An Introduction to Enzymes
- Much of the history of biochemistry is the history of enzyme research, Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach.
- Research continued in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts.
- In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by "ferments."
- He postulated that these ferments were in- separable from the structure of living yeast cells; this view, called vitalism, prevailed for decades.
- Then in 1897 Eduard Buchner discovered that yeast extracts could ferment, sugar to alcohol, proving that fermentation was promoted by molecules that continued to function when removed from cells
- Buchner's experiment at once marked the end of vitalistic notions and the dawn of the science of biochemistry.
- Frederick W. Kühne later gave the name enzymes to the molecules detected by Buchner.
- The isolation and crystallization of urease by James Sumner in 1926 was a breakthrough in early enzyme studies.
- Summer found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins.
- In the absence of other examples, this idea remained controversial for some time.
- Only in the 1930s was Sumner's conclusion widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins.
- During this period, J. B. S. Haldane wrote a treatise entitled Enzymes.
- Although the molecular nature of enzymes was not yet fully appreciated, Haldane made the remarkable suggestion that weak bonding interactions between an enzyme and its substrate might be. used to catalyze a reaction.
- This might lies at the heart of our current understanding of enzymatic catalysis.
- Since the latter part of the twentieth century, thousands of enzymes have been purified, their structures Can elucidated, and their mechanisms explained.
Most Enzymes Are Proteins
With the exception of a small group of catalytic RNA molecules (Chapter 26), all enzymes are proteins.
Their catalytic activity depends on the integrity of their native protein conformation.
If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost.
If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed
This the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity.
Enzymes, like other proteins, have molecular weights ranging from about 12,000 to more than 1 million.
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 Fe, Mg, Mr, or Zn (Table 6-1), or a complex organic or metallo organic molecule called a coenzyme.
Coenzymes act as transient (Table 6-2).
Most are derived from vitamins, organic nutrients required in small amounts in the diet.
We consider coenzymes in more detail as we encounter them in the metabolic pathways discussed in Part II
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.
Finally, some enzyme proteins are modified covalently by phosphorylation, glycosylation, and other processes.
Many of these alterations are involved in the regulation of enzyme activity
Enzymes Are Classified by the Reactions They Catalyze
Many enzymes have been named by adding the suffix "ase" to the name of their substrate or to a word or phrase describing their activity.
Thus urease catalyzes hydrolysis of urea, and DNA polymerase catalyzes the polymerization of nucleotides to form DNA.
Other enzymes were named by their discoverers for a broad function, before the specific reaction catalyzed was known,
For example, an enzyme known to act in the digestion of foods was named pepain, from the Greek pepsis, "digestion." and lysozyme was named for its ability to lyse (break down) bacterial cell walls.
Still others were named for their source: trypsin, named in part from the Greek trypsin, "to wear down," was obtained by nabbing pancreatic tissue with glycerin.
Sometimes the same enzyme has two or more names, or two different enzymes have the same name.
Because of such ambiguities, and the ever-increasing number of newly discovered enzymes, biochemists, by interational agreement, have adopted a system for naming and classifying enzymes
This system divides enzymes into six classes, each with subclasses, based on the type of reaction catalyzed (Table 6-3).
Each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it, catalyse.
As an example, the formal systematic name of the enzyme catalyzing the reaction class (phosphotransferase);
the third number (1), a phosphotransferase with a hydroxyl group as acceptor and the fourth number (1), -glucose as the phosphoryl group acceptor
For many enzymes, a common name is more frequently used in this case hexokinase.
A complete list and description of the thousands of known enzymes is maintained by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
(www.chem.quid.ac.uk/ibmb/enzyme).
This chapter is devoted primarily to principles and properties common to all enzymes
SUMMARY 6.1 An Introduction to Enzymes
Life depends on powerful and specific catalysts the enzymes. Almost every biochemical reaction is catalyzed by an enzyme.
With the exception of a few catalytic RNAs, all known enzymes are proteins.
Many require nonprotein coenzymes or cofactors for their catalytic function.
ATP D-glucose-ADP+ D-gluse 6-phosphate
is ATP ghase phosphotransferase, which indicates that it catalyzes the transfer of a phosphoryl group from ATP to glucose
It Ergyme Commission number (E.C. mum. ber) is 2.7.1.1. The first number (2) denotes the class name (transferase); the second number (7), the sub
Enzymes are classified according to the type of reaction they catalyze. All enzymes have formal
E.C. numbers and names, and most have trivial
To understand catalysis, we must first appreciate, the important distinction between reaction equilibria and reaction rates.
The function of a catalyst is to increase the rate of a reaction.
Catalysts do not affect reaction equilibria.
Any reaction, such as SP, can be described by a reaction coordinate diagram (Fig. 6-2), a picture of the energy changes during the reaction.
As discussed in Chapter 1, energy in biological systems is described in terms of free energy, G.
In the coordinate diagram, the free energy of the system is plotted against.
the progress of the reaction (the reaction coordinate).
The starting point for either the forward or the reverse reaction is called the ground state, the contribution to the free energy of the system by an average molecule (S or P) under a given set of conditions.
AGURE 6-1 inding of a substrate to an mayme at the active site. The enzyme dymotrypsin, with bound substrate in red (PDB ID 70CH Some key activeste amino acid residues appear as a red splotchun the ymme surface.
6.2 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, Further more, 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 two or more molecules in the precise orientation required for reaction.
Reactions required to digest food, send nerve signals, or contract a misele simply do not occur at a useful rate without catalysis
An enzyme circumvents these problems by providing a specific environment within 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 grups 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, whose existence was 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 behaviour of enzyme catalyzed reactions and for theoretical descriptions of enzyme mechanisms.
Enzymes Affect Reaction Rates, Not Equilibria
A simple enzymatic reaction might be written
KEY CONVENTION:
To describe the free-energy changes for reactions, chemists define a standand set of conditions (temperature 208 K; partial pressure of each gas 1 atm, or 101.3 kPa concentration of each solute I s) and express the free energy change for a reacting system under these conditions as AG", the standard free-energy change.
Because biochemical systems commonly involve H concentrations far below 1 M, biochemists define a biochemical standard free-energy change, ∆G, the standard free-energy change at pH 7.0, we employ this definition throughout the book.
The equilibrium between S and P reflects the difference in the free energies of their ground states.
In the example shown in Figure 6-2, the free energy of the ground state of P is lower than that of S, so AG for the reaction is negative and the equilibrium favors P.
The position and direction of equilibrium are not affected by any catalyst.
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the appropriate enzyme is present, because the rate of the reaction is increased. This general principle is illustrated in the conver sion of sucrose and oxygen to carbon dioxide and water:
A favorable equilibrium does not mean that the SP conversion will occur at a detectable rate. The rate of a reaction is dependent on an entirely different parameter. There is an energy barrier between S and P the energy required for alignment of reacting groups, formation of transient unstable charges, bond re arrangements, and other transformations required for the reaction to proceed in either direction. This is illus trated by the energy "hill" in Figures 6-2 and 6-3, Tb undergo reaction, the molecules must overcome this barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which de cay to the S or P state is equally probable (it is downhill either way). This is called the transition state. The transition state is not a chemical species with any signif icant stability and should not be confused with a reac tion intermediate (such as ES or EP). It is simply a fleeting molecular moment in which events such as bond breakage, band formation, and charge development have proceeded to the precise point at which decay to either substrate or product is equally likely. The stiffer ence between the energy levels of the ground state and the transition state is the activation energy AG The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction Reaction rates can be increased by mising the tempera ture and/or pressure, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier. Alternatively, the activation energy can be low ered by adding a catalyst (Fig. 6-3). Catalysts enhance maction rates by lowering activation energies
Enzymes are no exception to the rule that catalysts do not affect reaction equilibria. The bidirectional ar rows in Equation 6-1 nuke this point: any enzyme that catalyzes the reaction S-P also catalyzes the reaction PS. The role of enzymes is to accelerate the inter conversion of Sand P. The enzyme is not used up in the process, and the equilibrium point is unaffected. How ever, the reaction reaches equilibrium much faster when
Transition state (1) A
اد
Reaction coordinate
FIGURE 6-3 Reaction coordinate diagram comparing enzyme
catalyzed and uncatalyzed reactions in the reactions→P the 15 and EP intermediates occipy mnima in the energy progress curve of the enzyme-catalyzed reaction. The tems AG and AC comespond to the activation energy ker the uncatalyad reaction and the overall activation ausgy for the catalyzed seactkin, respectively. The atha bon energy is lowerwhen the enzyme catalyzes the reaction
CulO 120, 1200, +11H₂O
This conversion, which takes place through a series of separate reactions, has a very large and negative AG and at equilibrium the amount of sucrose present is neg ligible. Yet sucrose is a stable compound, because the activation energy barrier that must be overcome before. sucrose reacts with oxygen is quite high. Sucrose can be red in a container with oxygen almost indefinitely without reacting. In cells, however, sucrose is readily broken down to CO, and HO in a series of reactions cal alyzed by enzymes. These enzymes not only accelerate the reactions, they organize and control them so that much of the energy released is recovered in other chem ical forms and made available to the cell for other tasks, The reaction pathway by which sucrose (and other sug ars) is broken down is the primary energy-yielding path way for cells, and the enzymes of this pathway allow the reaction sequence to proceed on a biologically useful time scale
Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction intermediates A reaction intermediate is any species on the reaction pathway that has a finite. chemical lifetime (longer than a molecular vibration, -10 seconds). When the SP reaction is catalyzed i by an enzyme, the ES and EP complexes can be consid ered intermediates, even though S and P are stable chemical species (Ean 6-1); the ES and EP complexes occupy valleys in the reaction coordinate diagram (Fig. 6-3). Additional, less stable chemical intermediates of ten exist in the course of an eryme-catalyzed reaction. The interconversion of two sequential reaction interme diates thus constitutes a reaction step. When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation en ergy, this is called the rate-limiting step in a simple case, the rate limiting step is the highest-energy point in the diagram for interconversion of S and P. In practice, the tate-limiting step can vary with reaction conditions, and for many enzymes several steps may have similar activation energies, which means they are all partially mate-limiting.
Activation energies are energy barriers to chemical reactions. These barriers are crucial to life itself. The rate - at which a molecule undergoes a particular reaction
In this chapter, lediterinerler to chemical species in the main pathway of a side eye-cataly reaction. In the c it of mutable pothwaysinlingmay ays (discussed in Part II), these term ured somewhat diffently. An entire uzymatic mation often referred to as a step in a pathway, and the prodad cainis (which is the sidstnite for the next eve in the pathway) is referred to an intermediate