Enzyme Kinetics
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Wikipedia-Article "Enzyme Kinetics"
Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Because of their role in maintaining life processes, the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics. The macromolecular components of almost all enzymes are composed of protein, except for a class of RNA modifying catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other RNAs. Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals, and enzymes of the circulatory system are responsible for regulating the clotting of blood. Almost every significant life process is dependent on enzyme activity.
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Enzyme Classifications
Traditionally, enzymes were simply assigned names by the investigator who discovered the enzyme. As knowledge expanded, systems of enzyme classification became more comprehensive and complex. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.). These rules give each enzyme a unique number. The I.U.B. system also specifies a textual name for each enzyme. The enzyme's name is comprised of the names of the substrate(s), the product(s) and the enzyme's functional class. Because many enzymes, such as alcohol dehydrogenase, are widely known in the scientific community by their common names, the change to I.U.B.-approved nomenclature has been slow. In everyday usage, most enzymes are still called by their common name. Enzymes are also classified on the basis of their composition. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small organic molecule. Complex enzymes are also known as holoenzymes. In this terminology the protein component is known as the apoenzyme, while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis.
Role of Coenzymes
The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD; or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate. Since coenzymes are chemically changed as a consequence of enzyme action, it is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated.
Enzyme Relative to Substrate Type
Although enzymes are highly specific for the kind of reaction they catalyze, the same is not always true of substrates they attack. For example, while succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid, alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction reactions but attacks a number of different alcohols, ranging from methanol to butanol. Generally, enzymes having broad substrate specificity are most active against one particular substrate. In the case of ADH, ethanol is the preferred substrate. Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. The enzymes known as racemases provide a striking exception to these generalities; in fact, the role of racemases is to convert D isomers to L isomers and vice versa. Thus racemases attack both D and L forms of their substrate. As enzymes have a more or less broad range of substrate specificity, it follows that a given substrate may be acted on by a number of different enzymes, each of which uses the same substrate(s) and produces the same product(s). The individual members of a set of enzymes sharing such characteristics are known as isozymes. These are the products of genes that vary only slightly; often, various isozymes of a group are expressed in different tissues of the body. The best studied set of isozymes is the lactate dehydrogenase (LDH) system. LDH is a tetrameric enzyme composed of all possible arrangements of two different protein subunits; the subunits are known as H (for heart) and M (for skeletal muscle). These subunits combine in various combinations leading to 5 distinct isozymes. The all H isozyme is characteristic of that from heart tissue, and the all M isozyme is typically found in skeletal muscle and liver. These isozymes all catalyze the same chemical reaction, but they exhibit differing degrees of efficiency. The detection of specific LDH isozymes in the blood is highly diagnostic of tissue damage such as occurs during cardiac infarct.
Enzyme-Substrate Interactions
The favored model of enzyme substrate interaction is known as the induced fit model. This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. After binding takes place, one or more mechanisms of catalysis generates transition- state complexes and reaction products. The possible mechanisms of catalysis are four in number:
Catalysis by Bond Strain
In this form of catalysis, the induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the transition state. The new conformation often forces substrate atoms and bulky catalytic groups, such as aspartate and glutamate, into conformations that strain existing substrate bonds
Catalysis by Proximity and Orientation
Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another. In addition to inducing strain, groups such as aspartate are frequently chemically reactive as well, and their proximity and orientation toward the substrate thus favors their participation in catalysis.
Catalysis Involving Proton Donors (Acids) and Acceptors (Bases)
Other mechanisms also contribute significantly to the completion of catalytic events initiated by a strain mechanism, for example, the use of glutamate as a general acid catalyst (proton donor).
Covalent Catalysis
In catalysis that takes place by covalent mechanisms, the substrate is oriented to active sites on the enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. One of the best-known examples of this mechanism is that involving proteolysis by serine proteases, which include both digestive enzymes (trypsin, chymotrypsin, and elastase) and several enzymes of the blood clotting cascade. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond, thereby causing hydrolysis of the peptide bond.
Chemical Reactions and Rates
According to the conventions of biochemistry, the rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into product(s) in a specified time period. Reaction rate is always dependent on the concentration of the chemicals involved in the process and on rate constants that are characteristic of the reaction. For example, the reaction in which A is converted to B is written as follows:
A ------> B
The rate of this reaction is expressed algebraically as either a decrease in the concentration of reactant A:
-[A] = k[B]
or an increase in the concentration of product B:
[B] = k[A]
In the second equation (of the 3 above) the negative sign signifies a decrease in concentration of A as the reaction progresses, brackets define concentration in molarity and the k is known as a rate constant. Rate constants are simply proportionality constants that provide a quantitative connection between chemical concentrations and reaction rates. Each chemical reaction has characteristic values for its rate constants; these in turn directly relate to the equilibrium constant for that reaction. Thus, reaction can be rewritten as an equilibrium expression in order to show the relationship between reaction rates, rate constants and the equilibrium constant for this simple case. The rate constant for the forward reaction is defined as k+1 and the reverse as k-1. At equilibrium the rate (v) of the forward reaction (A -----> B) is--- by definition--- equal to that of the reverse or back reaction (B -----> A), a relationship which is algebraically symbolized as:
(v)forward = (v)reverse
where, for the forward reaction:
vforward = k+1[A]
and for the reverse reaction:
vreverse = k-1[B]
In the above equations, k+1 and k-1 represent rate constants for the forward and reverse reactions, respectively. The negative subscript refers only to a reverse reaction, not to an actual negative value for the constant. To put the relationships of the two equations into words, we state that the rate of the forward reaction [vforward] is equal to the product of the forward rate constant k+1 and the molar concentration of A. The rate of the reverse reaction is equal to the product of the reverse rate constant k-1 and the molar concentration of B. At equilibrium, the rate of the forward reaction is equal to the rate of the reverse reaction leading to the equilibrium constant of the reaction and is expressed by:
[B]/[A] = k+1/k-1 = Keq
This equation demonstrates that the equilibrium constant for a chemical reaction is not only equal to the equilibrium ratio of product and reactant concentrations, but is also equal to the ratio of the characteristic rate constants of the reaction.