Introduction to Enzymes

The following has been excerpted from a very popular Worthington publication which was originally published in 1972 as the Manual of Clinical Enzyme Measurements. While some of the presentation may seem somewhat dated, the basic concepts are still helpful for researchers who must use enzymes but who have little background in enzymology.

Introduction
The use of enzymes in the diagnosis of disease is one of the important benefits derived from the intensive research in biochemistry since the 1940's. Enzymes have provided the basis for the field of clinical chemistry. It is, however, only within the recent past few decades that interest in diagnostic enzymology has multiplied. Many methods currently on record in the literature are not in wide use, and there are still large areas of medical research in which the diagnostic potential of enzyme reactions has not been explored at all. This section has been prepared by Worthington Biochemical Corporation as a practical introduction to enzymology. Because of its close involvement over the years in the theoretical as well as the practical aspects of enzymology, Worthington's knowledge covers a broad spectrum of the subject. Some of this information has been assembled here for the benefit of laboratory personnel. This section summarizes in simple terms the basic theories of enzymology.

I. Enzymes and Life Processes

The living cell is the site of tremendous biochemical activity called metabolism. This is the process of chemical and physical change which goes on continually in the living organism. Build-up of new tissue, replacement of old tissue, conversion of food to energy, disposal of waste materials, reproduction - all the activities that we characterize as "life." This building up and tearing down takes place in the face of an apparent paradox. The greatest majority of these biochemical reactions do not take place spontaneously. The phenomenon of catalysis makes possible biochemical reactions necessary for all life processes. Catalysis is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change. The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism. The oxidation of a fatty acid to carbon dioxide and water is not a gentle process in a test tube - extremes of pH, high temperatures and corrosive chemicals are required. Yet in the body, such a reaction takes place smoothly and rapidly within a narrow range of pH and temperature. In the laboratory, the average protein must be boiled for about 24 hours in a 20% HCl solution to achieve a complete breakdown. In the body, the breakdown takes place

in four hours or less under conditions of mild physiological temperature and pH. It is through attempts at understanding more about enzyme catalysts - what they are, what they do, and how they do it - that many advances in medicine and the life sciences have been brought about.

Early Enzyme Discoveries

The existence of enzymes has been known for well over a century. Some of the earliest studies were performed in 1835 by the Swedish chemist Jon Jakob Berzelius who termed their chemical action catalytic. It was not until 1926, however, that the first enzyme was obtained in pure form, a feat accomplished by James B. Sumner of Cornell University. Sumner was able to isolate and crystallize the enzyme urease from the jack bean. His work was to earn him the 1947 Nobel Prize. John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical Research shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used to crystallize several enzymes.

Chemical Nature of Enzymes

All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. See Figure 1.

Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000.

Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ionactivator) is called the holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

According to Holum, the cofactor may be: 1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely attached to the protein part. 2. A prosthetic group - an organic substance which is dialyzable and thermostable which is firmly attached to the protein or apoenzyme portion. 3. A metal-ion-activator - these include K +, Fe ++, Fe+ + +, Cu ++, Co ++, Zn + +, Mn + +, Mg + +, Ca ++, and Mo + + + .

Specificity of Enzymes
One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze. A few enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are four distinct types of specificity: 1. Absolute specificity - the enzyme will catalyze only one reaction. 2. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. 3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

Though enzymes exhibit great degrees of specificity, cofactors may serve many apoenzymes. For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a great number of dehydrogenase reactions in which it acts as a hydrogen acceptor. Among them are the alcohol dehydrogenase, malate dehydrogenase and lactate dehydrogenase reactions.

Naming and Classification

Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin, most enzyme names end in "ase". The International Union of Biochemistry (I.U.B.) initiated standards of enzyme nomenclature which recommend that enzyme names indicate both the substrate acted upon and the type of reaction catalyzed. Under this system, the enzyme uricase is called urate: O2 oxidoreductase, while the enzyme glutamic oxaloacetic transaminase (GOT) is called L-aspartate: 2-oxoglutarate aminotransferase. Enzymes can be classified by the kind of chemical reaction catalyzed. I. Addition or removal of water A. Hydrolases - these include esterases, carbohydrases, nucleases, deaminases, amidases, and proteases B. Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase II. Transfer of electrons A. Oxidases B. Dehydrogenases III. Transfer of a radical A. Transglycosidases - of monosaccharides B. Transphosphorylases and phosphomutases - of a phosphate group C. Transaminases - of amino group D. Transmethylases - of a methyl group E. Transacetylases - of an acetyl group IV. Splitting or forming a C-C bond A. Desmolases V. Changing geometry or structure of a molecule A. Isomerases VI. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other tri-phosphate A. Ligases

II. Enzyme Kinetics

Basic Enzyme Reactions
Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. The basic enzymatic reaction can be represented as follows

where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction.

Energy Levels
Chemists have known for almost a century that for most chemical reactions to proceed, some form of energy is needed. They have termed this quantity of energy, "the energy of activation." It is the magnitude of the activation energy which determines just how fast the reaction will proceed. It is believed that enzymes lower the activation energy for the reaction they are catalyzing. Figure 3 illustrates this concept.

The enzyme is thought to reduce the "path" of the reaction. This shortened path would require less energy for each molecule of substrate converted to product. Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present (the shortened "path") than when it is absent. Hence, the reaction is said to go faster in a given period of time.

The Enzyme Substrate Complex

A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate substance which is known as the enzyme substrate complex. The reaction can be represented as:

If this reaction is combined with the original reaction equation [1], the following results:

The existence of an intermediate enzyme-substrate complex has been demonstrated in the laboratory, for example, using catalase and a hydrogen peroxide derivative. At Yale University, Kurt G. Stern observed spectral shifts in catalase as the reaction it catalyzed proceeded. This experimental evidence indicates that the enzyme first unites in some way with the substrate and then returns to its original form after the reaction is concluded.

Chemical Equilibrium
The study of a large number of chemical reactions reveals that most do not go to true completion. This is likewise true of enzymatically-catalyzed reactions. This is due to the reversibility of most reactions. In general:

where K +1 is the forward reaction rate constant and K-1 is the rate constant for the reverse reaction. Combining the two reactions gives:

Applying this general relationship to enzymatic reactions allows the equation:

Equilbrium, a steady state condition, is reached when the forward reaction rates equal the

backward rates. This is the basic equation upon which most enzyme activity studies are based.

Factors Affecting Enzyme Activity

Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme analysis. The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

Enzyme Concentration
In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as:

These reactions are said to be "zero order" because the rates are independent of substrate concentration, and are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the amount of product.

Table I Reaction Orders with Respect to Substrate Concentration Order zero first second Rate Equation rate = k rate = k[S] rate = k[S] [S]=k[S]2 Comments rate is independent of substrate concentration rate is proportional to the first power of substrate concentration rate is proportional to the square of the substrate concentration rate is proportional to the first power of each of two reactants second rate = k[S1][S2]

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order. In Figure 5, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme activity is generally greatest when substrate concentration is unlimiting.

When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 6.

Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first order between B and C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve where the reaction is zero order. A reaction is most likely to be zero order initially since substrate concentration is then highest. To be certain that a reaction is zero order, multiple measurements of product (or substrate) concentration must be made. Figure 7 illustrates three types of reactions which might be encountered in enzyme assays and shows the problems which might be enountered if only single measurements are made. B is a straight line representing a zero order reaction which permits accurate determination of enzyme activity for part or all of the reaction time. A represents the type of reaction that was shown in Figure 6. This reaction is zero order initially and then slows, presumably due to substrate exhaustion or product inhibition. This type of reaction is sometimes referred to as a "leading" reaction. True "potential" activity is represented by the dotted line. Curve C represents a reaction with an initial "lag" phase. Again the dotted line represents the potentially measurable activity. Multiple determinations of product concentration enable each curve to be plotted and true activity determined. A single end point determination at E would lead to the false conclusion that all three samples had identical enzyme concentration.

Substrate Concentration
It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). This is represented graphically in Figure 8.

It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this maximum velocity and equation (7),

Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is shown in Figure 8. Using this constant and the fact that Km can also be defined as:

K +1, K-1 and K +2 being the rate constants from equation (7). Michaelis developed the following expression for the reaction velocity in terms of this constant and the substrate concentration.

Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme. 1. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. 2. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.
3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently

assumed to be enzyme's natural substrate, though this is not true for all enzymes.

Effects of Inhibitors on Enzyme Activity

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition. Most theories concerning inhibition mechanisms are based on the existence of the enzymesubstrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory.

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs.

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally. Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 10.

Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the reaction velocity decreasing after the maximum velocity has been reached.

Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them.

This causes the reaction rate to drop since all of the enzyme present is not being used.

Temperature Effects
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in Figure 13, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40C, most enzyme determinations are carried out somewhat below that temperature. Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5C or below is generally the most suitable. Some enzymes lose their activity when frozen.

Effects of pH
Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. This is graphically illustrated in Figure 14.

Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. The optimum pH value will vary greatly from one enzyme to another, as Table II shows:

Table II pH for Optimum Activity Enzyme Lipase (pancreas) Lipase (stomach) Lipase (castor oil) Pepsin Trypsin Urease Invertase Maltase Amylase (pancreas) Amylase (malt) Catalase

pH Optimum 8.0 4.0 - 5.0 4.7 1.5 - 1.6 7.8 - 8.7 7.0 4.5 6.1 - 6.8 6.7 - 7.0 4.6 - 5.2 7.0

In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible.

References 1. Bennett, T. P., and Frieden, E.: Modern Topics in Biochemistry, pg. 43-45, Macmillan, London (1969). 2. Holum, J.: Elements of General and Biological Chemistry, 2nd ed., 377, Wiley, NY (1968).

3. Martinek, R.: Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969). 4. Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958). 5. Pfeiffer, J.: Enzymes, the Physics and Chemistry of Life, pg 171-173, Simon and Schuster, NY (1954)