1、The Structure and Function of Enzymes Chemical reactions in biological systems rarely occur in the absence of a catalyst. These catalysts are specific proteins called enzymes. The striking characteristics of all enzymes are their catalytic power and specificity. Furthermore, the activity of many enz
2、ymes is regulated. In addition, some enzymes are intimately involved in the transformation of different forms of energy. Let us examine these highly distinctive and biologically crucial properties of enzymes. Enzymes Have Enormous Catalytic Power Enzymes accelerate reactions by factors of at least a
3、 million. Indeed, most reactions in biological systems do not occur at perceptible rates in the absence of enzymes. Even a reaction as simple as the hydration of carbon dioxide is catalyzed by an enzyme. H2CO3 CO2+ H2O Otherwise, the transfer of CO2 from the tissues into the blood and then to the al
4、veolar air would be incomplete. Carbonic anhydrase, the enzyme that catalyzes this reaction, is one of the fastest known. Each enzyme molecule can hydrate 105 molecules of CO2 in one second. This catalyzed reaction is 107 times faster than the uncatalyzed reaction. Enzymes are Highly Specific Enzyme
5、s are highly specific both in the reaction catalyzed and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. The degree of specificity for substrate is usually high and sometimes virtually absolute.
6、Let us consider proteolytic enzymes as an example. The reaction catalyzed by these enzymes is the hydrolysis of a peptide bond. N H C H R1 C O N H C H R2 C O + H2O Peptide N H C H R1 C O -H3N C H R2 C O + O- Carboxyl componentAmino component Most proteolytic enzymes also catalyze a different but rel
7、ated reaction, namely the hydrolysis of an ester bond. R1C O OR2+ H2OR1C O O- +HOR2+ H+ Ester AcidAlcohol Proteolytic enzymes vary markedly in their degree of substrate specificity. Subtilisin, which comes from certain bacteria, is quite undiscriminating about the nature of the side chains adjacent
8、to the peptide bond to be cleaved. Trypsin is quite specific in that it splits peptide bonds on the carboxyl side of lysine and argentine residues only. Thrombin, an enzyme participating in blood clotting, is even more specific than trypsin. The side chain on the carboxyl side of the susceptible pep
9、tide bond must be arginine, whereas the one on the amino side must be glycine. Another example of the high degree of specificity of enzymes is provided by DNA polymerase I. This enzyme synthesizes DNA by linking together four kinds of nucleotide building blocks. The sequence of nucleotides in the DN
10、A strand that is being synthesized is determined by the sequence of nucleotides in another DNA strand that serves as a template. DNA polymerase I is remarkably precise in carrying out the instructions given by the template. The wrong nucleotide is inserted into a new DAN strand less than once in a m
11、illion times. The Activities of Some Enzymes Are Regulated Some enzymes are synthesized in an inactive precursor form and are activated at a physiologically appropriate time and place. The digestive enzymes exemplify this kind of control. For example, trypsinogen is synthesized in the pancreas and i
12、s activated by peptide-bond cleavage in the small intestine to form the active enzyme trypsin. This type of control is also repeatedly used in the sequence of enzymatic reactions leading to the clotting of blood. The enzymatically inactive precursors of proteolytic enzymes are called zymogens. Anoth
13、er mechanism that controls activity is the covalent insertion of a small group on an enzyme. This control mechanism is called covalent modification. For example, the activities of the enzymes that synthesize and degrade glycogen are regulated by the attachment of a phosphoryl group to a specific ser
14、ine residue on these enzymes. This modification can be reversed by hydrolysis. Specific enzymes catalyze the insertion and removal of phosphoryl and other modifying groups. A different kind of regulatory mechanism affects many reaction sequences resulting in the synthesis of small molecules such as
15、amino acids. The enzyme that catalyzes the first step in such a biosynthetic pathway is inhibited by the ultimate product. The biosynthesis of isoleucine in bacteria illustrates this type of control, which is called feedback inhibition. Threonine is converted into isoleucine in five steps, the first
16、 of which is catalyzed by threonine deaminase. This enzyme is inhibited when the concentration of isoleucine reaches a sufficiently high level. Isoleucine binds to a regulatory site on the enzyme, which is distinct from its catalytic site. The inhibition of threonine deasminase is mediated by an all
17、osteric interaction, which is reversible. When the level of isoleucine drops sufficiently, threonine deaminase becomes active again, and consequently isoleucine is again synthesized. The specificity of some enzymes is under physiological control. The synthesis of lactose by the mammary gland is a pa
18、rticularly striking example. Lactose synthetase, the enzyme that catalyzes the synthesis of lactose, consists of a catalytic subunit and a modifier subunit. The catalytic subunit by itself cannot synthesize lactose. It has a different role, which is to catalyze the attachment of galactose to a prote
19、in that contains a covalently linked carbohydrate chain. The modifier subunit alters the specificity of the catalytic subunit so that it links galactose to glucose to form lactose. The level of the modifier subunit is under hormonal control. During pregnancy, the catalytic subunit is formed in the m
20、ammary gland, but little modifier subunit is formed. At the time of birth, hormonal levels change drastically, and the modifier subunit is synthesized in large amounts. The modifier subunit then binds to the catalytic subunit to form an active lactose synthetase complex that produces large amounts o
21、f lactose. This system clearly shows that hormones can exert their physiological effects by altering the specificity of enzymes. Enzymes Transform Different Kinds of Energy In many biochemical reactions, the energy of the reactants is converted into a different form with high efficiency. For example
22、, in photosynthesis, light energy is converted into chemical-bond energy. In mitochondria, the free energy contained in small molecules derived from foods is converted into a different currency, that of adenosine triphosphate (ATP). The chemical-bond energy of ATP is them utilized in many different
23、ways. In muscular contraction, the energy of ATP is converted into mechanical energy. Cells and organelles have pumps that utilize ATP to transport molecules and ions against chemical and electrical gradients. These transformations of energy are carried out by enzyme molecules that are integral part
24、s of highly organized assembilies. Enzymes Do Not Alter Reaction Equilibria An enzyme is a catalyst and consequently it cannot alter the equilibrium of a chemical reaction. This means that an enzyme accelerates the forward and reverse reaction by precisely the same factor. Consider the interconversi
25、on of A and B. Suppose that in the absence of enzyme the forward rate (KF) is 10-4sec-1and the reverse rate (KR) is 10-6sec-1. The equilibrium constant K is given by the ratio of these rates: A 10-4sec-1 10-6sec-1 B K= B A = KF KR = 10-4 10-6 = 100 The equilibrium concentration of B is 100 times tha
26、t of A, whether or not enzyme is present. However, it would take several hours to approach this equilibrium without enzyme, whereas equilibrium would be attained within a second when enzyme is present. Thus, enzymes accelerate the attainment of equilibria but do not shift their positions. Enzymes De
27、crease the Activation Energies of Reactions Catalyzed by Them A chemical reaction, AB, goes through a transition state that has a higher energy than either A or B. The rate of the forward reaction depends on the temperature and on the difference in free energy between that of A and the transition st
28、ate, which is called the Gibbs free energy of a activation and symbolized by G. G G transition state-Gsubstrate The reaction rate is proportional to the fraction of molecules that have a free energy equal to or greater than G . The proportion of molecules that have an energy equal to or greater than
29、 G increases with temperature. Enzymes accelerate reactions by decreasing G , the activation barrier. The combination of substrate and enzyme creates a new reaction pathway whose transition-state energy is lower than it would be if the reaction were taking place in the absence of enzyme. Formation o
30、f an Enzyme-Substrate Complex Is the First Step in Enzymatic Catalysis The making and breaking of chemical bonds by an enzyme are preceded by the formation of an enzyme-substrate (ES) complex. The substrate is bound to a specific region of the enzyme called the active site. Most enzymes are highly s
31、elective in their binding of substrates. Indeed, the catalytic specificity of enzymes depends in large part on the specificity of the binding process. Furthermore, the control of enzymatic activity may also take place at this stage. The existence of ES complexes has been shown in a variety of ways:
32、ES complexes have been directly visualized by electron microscopy and X-ray crystallography. Complexes of nucleic acids and their polymerase enzymes are evident in electron micrographs. Detailed information concerning the location and interactions of glycyl-L-tyrosine, a substrate of carboxypeptidas
33、e A, has been obtained from X-ray studies of that ES complex. The physical properties of an enzyme, such as its solubility or heat stability, frequently change upon formation of an ES complex. The Spectroscopic characteristics of many enzymes and substrates change upon formation of an ES complex jus
34、t as the absorption spectrum of deoxyhemoglobin changes markedly when it binds oxygen or when it is oxidized to the ferric state, as described previously. These changes are particularly striking if the enzyme contains a colored prosthetic group. Tryptophan synthetase, a bacterial enzyme that contain
35、s a pyridoxal phosphate prosthetic group, affords a nice illustration. This enzyme catalyzes the synthesis of L-tryptophan from L-serine and indole. The addition of L-serine to the enzyme produces a marked increase in the fluorescence of the pyridoxal phosphate group. The subsequent addition of indo
36、le, the second substrate, quenches this fluorescence to a level lower than that of the enzyme alone. Thus, fluorescence spectroscopy reveals the existence of an enzyme-serine complex and of an enzyme-serine-indole complex. Other spectroscopic techniques, such as nuclear and electron magnetic resonan
37、ce, also are highly informative about ES interactions. A high degree of stereospecificity is displayed in the formation of ES complexes. For example, D-serine is not a substrate of tryptophan synthetase. Indeed, the D-isomer does not even bind to the enzyme. This implies that the substrate-binding s
38、ite has a very well defined shape. ES complexes can sometimes be isolated in pure form. For an enzyme that catalyzes the reaction A+B=C, it is sometimes possible to isolate an EA complex. This can be done if the enzyme has a sufficiently high affinity for A and if B is absent from the mixture. At a
39、constant concentration of enzyme, the reaction rate increases with increasing substrate concentration until a maximal velocity is reached. In contrast, uncatalyzed reactions do not show this saturation effect. In 1913, Leonor Michaelis interpreted the maximal velocity of an enzyme-catalyzed reaction
40、 in terms of the formation of a discrete ES complex. At a sufficiently high substrate concentration, the catalytic sites are filled and so the reaction rate reaches a maximum. This is the oldest and most general evidence for the existence of ES complexes. Some Features of Active Sites The active sit
41、e of an enzyme is the region that binds the substrates (and the prosthetic group, if any) and contributes the residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups. Although enzymes differ widely in structure, specificity, and mode of
42、catalysis, a number of generalizations concerning their active sites can be stated: The active site takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residues in an enzyme are not in contact with the substrate. This raises the intriguing question of why enzyme
43、s are so big. Nearly all enzymes are made up of more than 100 amino acid residues, which gives them a mass greater than 10 kdal and a diameter of more than 25. The active site is a three-dimensional entity. The active site of an enzyme is not a point, a line, or even a plane. It is an intricate thre
44、e-dimensional form made up of groups that come from different parts of the linear amino acid sequence-indeed, residues far apart in the linear sequence may interact more strongly than adjacent residues in the amino acid sequence, as has already been seen for myoglobin and hemoglobin. In lysozyme, an
45、d enzyme that will be discussed in more detail, the important groups in the active site are contributed by residues numbered 35,52,62,63, and 101 in the linear sequence of 129 amino acids. Substrates are bound to enzymes by relatively weak forces. ES complexes usually have equilibrium constants that
46、 range from 10-2 to10-8M, corresponding to free energies of interaction ranging from -3 to -12 kcal/mol. These values should be compared with the strengths of covalent bonds, which are between -50 and -110 kcal/mol. Active sties are clefts or crevices. In all enzymes of known structure, substrate mo
47、lecules are bound to a cleft or crevice from which water is usually excluded unless it is a reactant. The cleft also contains several polar residues that are essential for binding and catalysis. The nonpolar character of the cleft enhances the binding of substrate. In addition, the cleft creates a m
48、icroenvironment in which certain polar residues acquire special properties essential for their catalytic role. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. A substrate must have a matching shape to fit into the site. Emil Fischers metaphor of th
49、e lock and key, stated in 1894, has proved to be an essentially correct and highly fruitful way of looking at the stereospecificity of catalysis. However, recent work suggests that the active sites of some enzymes are not rigid. In such an enzyme, the shape of the active site is modified by the binding of substrate. The active site has a shape complementary to that of the substrate only after the substrate is bound. This process of dynamic recognition is called induced fit. Furthermore, some enzymes preferentially bind a strained from of the substrate corresponding to
