Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Enzyme Activities

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8Enzyme Activities

D. J. H. Shyu, J. T. C. Tzen, and C. L. Jeang

IntroductionFeatures of Enzymes

Most of the Enzymes are ProteinsChemical Composition of EnzymesEnzymes are SpecificEnzymes are RegulatedEnzymes are Powerful Catalysts

Enzymes and Activation EnergyEnzymes Lower the Activation EnergyHow Does an Enzyme work?

Enzyme Kinetics and MechanismRegulatory Enzymes

Feedback InhibitionNoncompetitive InhibitionCompetitive Inhibition

Enzyme KineticsData Presentation

Untransformed GraphicsLineweaver–Burk PlotsEadie–Hofstee PlotsHanes–Wolff PlotsEisenthal–Cornish-Bowden Plots (the Direct Linear

Plots)Hill Plot

Factors Affecting Enzyme ActivityEnzyme, Substrate, and Cofactor ConcentrationsEffects of pHEffects of Temperature

Methods Used in Enzyme AssaysGeneral ConsiderationsTypes of Assay MethodsDetection Methods

This manuscript was reviewed by Dr. Hsien-Yi Sung, Department of BiochemicalScience and Technology, National Taiwan University, Taipei, Taiwan, Republicof China, and Dr. Wai-Kit Nip, Department of Molecular Biosciences and Bio-engineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.

Spectrophotometric MethodsSpectrofluorometric MethodsRadiometric MethodsChromatographic MethodsElectrophoretic MethodsOther Methods

Selection of an Appropriate SubstrateUnit of Enzyme Activity

References

INTRODUCTIONLong before human history, our ancestors, chimpanzees, mighthave already experienced the mild drunk feeling of drinkingwine when they ate the fermented fruits that contained smallamounts of alcohol. Archaeologists have also found some sculp-tured signs on 8000-year-old plates describing the beer-makingprocesses. Chinese historians wrote about the lavish life of Tsou,a tyrant of Shang dynasty (around 1100 bc), who lived in a castlewith wine-storing pools. All these indicated that people graspedthe wine fermentation technique thousands of years ago. In theChin dynasty (around 220 bc) China, a spicy paste or sauce madefrom fermentation of soybean and/or wheat was mentioned. It isnow called soybean sauce.

Although people applied fermentation techniques and ob-served the changes from raw materials to special products, theydid not realize the mechanisms that caused the changes. Thismystery was uncovered by the development of biological sci-ences. Louis Pasteur claimed the existence and function of or-ganisms that were responsible for the changes. In the same era,Justus Liebig observed the digestion of meat with pepsin, asubstance found in stomach fluid, and proposed that a wholeorganism is not necessary for the process of fermentation.

In 1878, Kuhne was the first person to solve the conflict by in-troducing the word “enzyme,” which means “in yeast” in Greek.

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldra, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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168 Part 2: Biotechnology and Enzymology

This word emphasizes the materials inside or secreted by theorganisms to enhance the fermentation (changes of raw mate-rial). Buchner (1897) performed fermentation by using a cell-free extract from yeast of significantly high light, and thesewere molecules rather than lifeforms that did the work. In 1905,Harden and Young found that fermentation was accelerated bythe addition of some small dialyzable molecules to the cellfreeextract. The results indicated that both macromolecular and mi-cromolecular compounds are needed. However, the nature ofenzymes was not known at that time. A famous biochemist,Willstatter, was studying peroxidase for its high catalytic ef-ficiency; since he never got enough samples, even though thereaction obviously happened, he hesitated (denied) to concludethat the enzyme was a protein. Finally, in 1926, Summer pre-pared crystalline urease from jack beans, analyzed the propertiesof the pure compound, and drew the conclusion that enzymes areproteins. In the following years, crystalline forms of some pro-teases were also obtained by Northrop et al. The results agreedwith Summer’s conclusion.

The studies on enzyme behavior were progressing in paral-lel. In 1894, Emil Fischer proposed a “lock and key” theoryto describe the specificity and stereo relationship between anenzyme and its substrate. In 1902, Herri and Brown indepen-dently reported a saturation-type curve for enzyme reactions.They revealed an important concept in which the enzyme sub-strate complex was an obligate intermediate formed during theenzyme-catalyzed reaction. In 1913, Michaelis and Menten de-rived an equation describing quantitatively the saturation-likebehavior. At the same time, Monod and others studied the kinet-ics of regulatory enzymes and suggested a concerted model forthe enzyme reaction. In 1959, Fischer’s hypothesis was slightlymodified by Koshland. He proposed an induced-fit theory todescribe the moment the enzyme and substrate are attached,and suggested a sequential model for the action of allostericenzymes.

For the studies on enzyme structure, Sanger et al. were thefirst to announce the unveiling of the amino acid sequence ofa protein, insulin. After that, the primary sequences of somehydrolases with comparatively small molecular weights, suchas ribonuclease, chymotrypsin, lysozyme, and others, weredefined. Twenty years later, Sanger won his second NobelPrize for the establishment of the chain-termination reactionmethod for nucleotide sequencing of DNA. On the basis of thismethod, the deduction of the primary sequences of enzymesblossomed; to date, the primary structures of 55,410 enzymeshave been deduced. Combining genetic engineering technologyand modern computerized X-ray crystallography and/or Nucleaimagnetic resonance (NMR), about 15,000 proteins, including9268 proteins and 2324 enzymes, have been analyzed for theirthree-dimensional structures (Protein Data Bank (PDB): http://www.rcsb.org/pdb). Computer software was created, andprotein engineering on enzymes with demanded propertieswas carried on successfully (Fang and Ford 1998, Igarashiet al. 1999, Pechkova et al. 2003, Shiau et al. 2003, Swiss-PDBViewer(spdbv): http://www.expasy.ch/spdbv/mainpage.htm, SWISSMODELserver: http://www.expasy.org/swissmod/SWISS-MODEL.htm).

FEATURES OF ENZYMESMost of the Enzymes are Proteins

Proteins are susceptible to heat, strong acids and bases, heavymetals, and detergents. They are hydrolyzed to amino acids byheating in acidic solution and by the proteolytic action of en-zymes on peptide bonds. Enzymes give positive results on typi-cal protein tests, such as the Biuret, Millions, Hopkins-Cole, andSakaguchi reactions. X-ray crystallographic studies revealed thatthere are peptide bonds between adjacent amino acid residues inproteins. The majority of the enzymes fulfill the above criterion;therefore, they are proteins in nature. However, the catalytic el-ement of some well-known ribozymes is just RNAs in nature(Steitz and Moore 2003, Raj and Liu 2003).

Chemical Composition of Enzymes

For many enzymes, protein is not the only component requiredfor its full activity. On the basis of the chemical composi-tion of enzymes, they are categorized into several groups, asfollows:

1. Polypeptide, the only component, for example, lysozyme,trypsin, chymotrypsin, or papain.

2. Polypeptide plus one to two kinds of metal ions, forexample, α-amylase containing Ca2+, kinase contain-ing Mg2+, and superoxide dismutase having Cu2+ and/orZn2+.

3. Polypeptide plus a prosthetic group, for example, peroxi-dase containing a heme group.

4. Polypeptide plus a prosthetic group and a metal ion, forexample, cytochrome oxidase (a + a3) containing a hemegroup and Cu2+.

5. Polypeptide plus a coenzyme, for example, many dehydro-genases containing NAD+ or NADP+.

6. Combination of polypeptide, coenzyme, and a metal ion,for example, succinate dehydrogenase containing both theFAD and nonheme iron.

Enzymes are Specific

In the life cycle of a unicellular organism, thousands of reac-tions are carried out. For the multicellular higher organismswith tissues and organs, even more kinds of reactions are pro-gressing in every moment. Less than 1% of errors that occurin these reactions will cause accumulation of waste materials(Drake 1999), and sooner or later the organism will not be ableto tolerate these accumulated waste materials. These phenomenacan be explained by examining genetic diseases; for example,phenylketonuria (PKU) in humans, where the malfunction ofphenylalanine hydroxylase leads to an accumulation of metabo-lites such as phenylalanine and phenylacetate, and others, fi-nally causing death (Scriver 1995, Waters 2003). Therefore,enzymes catalyzing the reactions bear the responsibility for pro-ducing desired metabolites and keeping the metabolism goingsmoothly.

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8 Enzyme Activities 169

Enzymes have different types of specificities. They can begrouped into the following common types:

1. Absolute specificity: For example, urease (Sirko andBrodzik 2000) and carbonate anhydrase (Khalifah 2003)catalyze only the hydrolysis and cleavage of urea andcarbonic acid, respectively. Those enzymes having smallmolecules as substrates or that work on biosynthesis path-ways belong to this category.

2. Group specificity: For example, hexokinase prefers D-glucose, but it also acts on other hexoses (Cardenas et al.1998).

3. Stereo specificity: For example, d-amino acid oxidase re-acts only with d-amino acid, but not with l-amino acid;and succinate dehydrogenase reacts only with the fumarate(trans form), but not with maleate (cis form).

4. Bond specificity: For example, many digestive enzymes.They catalyze the hydrolysis of large molecules of foodcomponents, such as proteases, amylases, and lipases.They seem to have broad specificity in their substrates; forexample, trypsin acts on all kinds of denatured proteins inthe intestine, but prefers the basic amino acid residues atthe C-terminal of the peptide bond. This broad specificitybrings up an economic effect in organisms; they are notrequired to produce many digestive enzymes for all kindsof food components.

The following two less common types of enzyme specificityare impressive: (1) An unchiral compound, citric acid is formedby citrate synthase with the condensation of oxaloacetate andacetyl-CoA. An aconitase acts only on the moiety that comesfrom oxaloacetate. This phenomenon shows that, for aconitase,the citric acid acts as a chiral compound (Barry 1997). (2) Ifpeople notice the rare mutation that happens naturally, they willbe impressed by the fidelity of certain enzymes, for example,amino acyltRNA synthethase (Burbaum and Schimmel 1991,Cusack 1997), RNA polymerase (Kornberg 1996), and DNApolymerase (Goodman 1997); they even correct the accidentsthat occur during catalyzing reactions.

Enzymes are Regulated

The catalytic activity of enzymes is changeable under differentconditions. These enzymes catalyze the set steps of a metabolicpathway, and their activities are responsible for the states of cells.Intermediate metabolites serve as modulators on enzyme activ-ity; for example, at high concentrations of adenosine diphos-phate, the catalytic activity of the phosphofructokinase, pyruvatekinase, and pyruvate dehydrogenase complex are enhanced; asadenosine triphosphate becomes high, they will be inhibited.These modulators bind at allosteric sites on enzymes (Hammes2002). The structure and mechanism of allosteric regulatory en-zymes such as aspartate transcarbamylase (Cherfils et al. 1990)and ribonucleoside diphosphate reductase (Scott et al. 2001)have been well studied. However, allosteric regulation is notthe only way that the enzymatic activity is influenced. Covalentmodification by protein kinases (Langfort et al. 1998) and phos-phatases (Luan 2003) on enzymes will cause large fluctuations

in total enzymatic activity in the metabolism pool. In addition,sophisticated tuning phenomena on glycogen phosphorylase andglycogen synthase through phosphorylation and dephosphory-lation have been observed after hormone signaling (Nuttall et al.1988, Preiss and Romeo 1994).

Enzymes are Powerful Catalysts

The compound glucose will remain in a bottle for years withoutany detectable changes. However, when glucose is applied in aminimal medium as the only carbon source for the growth ofSalmonella typhimurium (or Escherichia coli), phosphorylationof this molecule to glucose-6-phosphate is the first chemicalreaction that occurs as it enters the cells. From then on, theactivated glucose not only serves as a fuel compound to beoxidized to produce chemical energy, but also goes throughnumerous reactions to become the carbon skeleton of variousmicro- and macrobiomolecules. To obtain each end product,multiple steps have to be carried out. It may take less than 30minutes for a generation to go through all the reactions. Onlythe existence of enzymes guarantees this quick utilization anddisappearance of glucose.

ENZYMES AND ACTIVATION ENERGYEnzymes Lower the Activation Energy

Enzymes are mostly protein catalysts; except for the presenceof a group of ribonucleic acid-mediated reactions, they are re-sponsible for the chemical reactions of metabolism in cells. Forthe catalysis of a reaction, the reactants involved in this reactionall require sufficient energy to cross the potential energy bar-rier, the activation energy (EA), for the breakage of the chemicalbonds and the start of the reaction. Few have enough energy tocross the reaction energy barrier until the reaction catalyst (theenzyme) forms a transition state with the reactants to lower theEA (Fig. 8.1). Thus, the enzyme lowers the barrier that usuallyprevents the chemical reaction from occurring and facilitates thereaction proceeding at a faster rate to approach equilibrium. Thesubstrates (the reactants), which are specific for the enzyme in-volved in the reaction, combine with enzyme to lower the EA ofthe reaction. One enzyme type will combine with one specifictype of substrate, that is, the active site of one particular enzymewill only fit one specific type of substrate, and this leads to theformation of and enzyme–substrate (ES) complex. Once the en-ergy barrier is overcome, the substrate is then changed to anotherchemical, the product. It should be noted that the enzyme itselfis not consumed or altered by the reaction, and that the overallfree-energy change (�G) or the related equilibrium also remainconstant. Only the activation energy of the uncatalyzed reaction(EAu) decreases to that of the enzyme-catalyzed reaction (EAc).

One of the most important mechanisms of the enzyme functionthat decreases the EA involves the initial binding of the enzymeto the substrate, reacting in the correct direction, and closing ofthe catalytic groups of the ES complex. The binding energy, partof the EA, is required for the enzyme to bind to the substrateand is determined primarily by the structure complementarities

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Figure 8.1. A transition state diagram, also called reactioncoordinate diagram, shows the activation energy profile of thecourse of an enzyme-catalyzed reaction. One curve depicts thecourse of the reaction in the absence of the enzyme, while the otherdepicts the course of reaction in the presence of the enzyme thatfacilitates the reaction. The difference in the level of free energybetween the beginning state (ground state) and the peak of thecurve (transition state) is the activation energy of the reaction. Thepresence of enzyme lowers the activation energy (EA) but does notchange the overall free energy (�G).

between the enzyme and the substrate. The binding energy isused to reduce the free energy of the transition-state ES complexbut not to form a stable, not easily separated, complex. Fora reaction to occur, the enzyme acts as a catalyst by efficientlybinding to its substrate, lowering the energy barrier, and allowingthe formation of product. The enzyme stabilizes the transitionstate of the catalyzed reaction, and the transition state is the rate-limiting state in a single-step reaction. In a two-step reaction,the step with the highest transition-state free energy is said tobe the rate-limiting state. Though the reaction rate is the speedat which the reaction proceeds toward equilibrium, the speed ofthe reaction does not affect the equilibrium point.

How Does an Enzyme work?

An enzyme carries out the reaction by temporarily combiningwith its specific kind of substrate, resulting in a slight changeof their structures to produce a very precise fit between the twomolecules. The introduction of strains into the enzyme and sub-strate shapes allows more binding energy to be available for thetransition state. Two models of this minor structure modificationare the induced fit, in which the binding energy is used to distortthe shape of the enzyme, and the induced strain, in which bind-ing energy is used to distort the shape of the substrate. Thesetwo models, in addition to a third model, the nonproductivebinding model, have been proposed to describe conformationalflexibility during the transition state. The chemical bonds of the

substrate break and form new ones, resulting in the formation ofnew product. The newly formed product is then released fromthe enzyme, and the enzyme combines with another substratefor the next reaction.

ENZYME KINETICS AND MECHANISMRegulatory Enzymes

For the efficient and precise control of metabolic pathways incells, the enzyme, which is responsible for a specific step in aserial reaction, must be regulated.

Feedback Inhibition

In living cells, a series of chemical reactions in a metabolic path-way occurs, and the resulting product of one reaction becomesthe substrate of the next reaction. Different enzymes are usu-ally responsible for each step of catalysis. The final product ofeach metabolic pathway will inhibit the earlier steps in the serialreactions. This is called feedback inhibition.

Noncompetitive Inhibition

An allosteric site is a specific location on the enzyme where anallosteric regulator, a regulatory molecule that does not directlyblock the active site of the enzyme, can bind. When the allostericregulator attaches to the specific site, it causes a change in theconformation of enzyme active site, and the substrate thereforewill not fit into the active site of enzyme; this results in the inhi-bition phenomenon. The enzyme cannot catalyze the reaction,and not only the amount of final product but also the concen-tration of the allosteric regulator decrease. Since the allostericregulator (the inhibitor) does not compete with the substrate forbinding to the active site of enzyme, the inhibition mechanismis called noncompetitive inhibition.

Competitive Inhibition

A regulatory molecule is not the substrate of the specific enzymebut shows affinity for attaching to the active site. When it occu-pies the active site, the substrate cannot bind to the enzyme forthe catalytic reaction, and the metabolic pathway is inhibited.Since the regulatory molecule will compete with the substratefor binding to the active site on enzyme, this inhibitory mecha-nism of the regulatory molecule is called competitive inhibition.

Enzyme Kinetics

During the course of an enzyme-catalyzed reaction, the plotof product formation over time (product formation profile) re-veals an initial rapid increase, approximately linear, of prod-uct, and then the rate of increase decreases to zero as timepasses (Fig. 8.2A). The slope of initial rate (vi or v), also calledthe steady state rate, appears to be initially linear in a plotwhere the product concentration versus reaction time follows the

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Figure 8.2. (A) Plot of progress curve during an enzyme-catalyzedreaction for product formation. (B) Plot of progress curves during anenzyme-catalyzed reaction for product formation with a differentstarting concentration of substrate. (C) Plot of reaction rate as afunction of substrate concentration measured from slopes of linesfrom (B).

establishment of the steady state of a reaction. When the reac-tion is approaching equilibrium or the substrate is depleted, therate of product formation decreases, and the slope strays awayfrom linearity. As is known by varying the conditions of thereaction, the kinetics of a reaction will appear linear during atime period in the early phase of the reaction, and the reactionrate is measured during this phase. When the substrate concen-trations are varied, the product formation profiles will displaylinear substrate dependency at this phase (Fig. 8.2B). The rates

for each substrate concentration are measured as the slope of aplot of product formation over time. A plot of initial rate as afunction of substrate concentration is demonstrated as shown inFigure 8.2C. Three distinct portions of the plot will be noticed: aninitial linear relationship showing first-order kinetic at low sub-strate concentrations; an intermediate portion showing curvedlinearity dependent on substrate concentration, and a final por-tion revealing no substrate concentration dependency, i.e., zero-order kinetic, at high substrate concentrations. The interpretationof the phenomenon can be described by the following scheme:

The initial rate will be proportional to the ES complex concentra-tion if the total enzyme concentration is constant and the substrateconcentration varies. The concentration of the ES complex willbe proportional to the substrate concentration at low substrateconcentrations, and the initial rate will show a linear relationshipand substrate concentration dependency. All of the enzyme willbe in the ES complex form when substrate concentration is high,and the rate will depend on the rate of ES transformation intoenzyme-product and the subsequent release of product. Addingmore substrate will not influence the rate, so the relationship ofrate versus substrate concentration will approach zero (Brown1902).

However, a lag phase in the progress of the reaction will benoticed in coupled assays due to slow or delayed response of thedetection machinery (see below). Though v can be determinedat any constant concentration of substrate, it is recommendedthat the value of substrate concentration approaching saturation(high substrate concentration) be used so that it can approach itslimiting value Vmax with greater sensitivity and prevent errorsoccurring at lower substrate concentrations.

Measurement of the rate of enzyme reaction as a function ofsubstrate concentration can provide information about the ki-netic parameters that characterize the reaction. Two parametersare important for most enzymes—Km, an approximate measure-ment of the tightness of binding of the substrate to the enzyme;and Vmax, the theoretical maximum velocity of the enzyme reac-tion. To calculate these parameters, it is necessary to measure theenzyme reaction rates at different concentrations of substrate, us-ing a variety of methods, and analyze the resulting data. For thestudy of single-substrate kinetics, one saturating concentrationof substrate in combination with varying substrate concentra-tions can be used to investigate the initial rate (v), the catalyticconstant (kcat), and the specific constant (kcat/Km). For inves-tigation of multisubstrate kinetics, however, the dependence ofv on the concentration of each substrate has to be determined,one after another; that is, by measuring the initial rate at vary-ing concentrations of one substrate with fixed concentrations ofother substrates.

A large number of enzyme-catalyzed reactions can be ex-plained by the Michaelis-Menten equation:

v = Vmax[S]

(Km + [S])= kcat[E][S]

(Km + [S]),

where [E] and [S] are the concentrations of enzyme and sub-strate, respectively (Michaelis and Menten 1913). The equationdescribes the rapid equilibrium that is established between thereactant molecules (E and S) and the ES complex, followed by

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slow formation of product and release of free enzyme. It assumesthat k2 � k−1 in the following equation, and Km is the value of[S] when v = Vmax/2:

E + Sk1

�k−1

ESk2−→ E + P.

Briggs and Haldane (1924) proposed another enzyme kineticmodel, called the steady state model; steady state refers to aperiod of time when the rate of formation of ES complex is thesame as rate of formation of product and release of enzyme. Theequation is commonly the same as that of Michaelis and Menten,but it does not require k2 � k−1, and Km = (k−1 + k2)/k1, be-cause [ES] now is dependent on the rate of formation of theES complex (k1) and the rate of dissociation of the ES com-plex (k−1 and k2). Only under the condition that k2 � k−1 willKs = k−1/k1 and be equivalent to Km, the dissociation constantof the ES complex {Ks = k−1/k1 = ([E] − [ES])[S]/[ES]}, oth-erwise Km > Ks.

The Km is the substrate concentration at half of the maximumrate of enzymatic reaction, and it is equal to the substrate con-centration at which half of the enzyme active sites are saturatedby the substrate in the steady state. So, Km is not a useful mea-sure of ES binding affinity in certain conditions when Km is notequivalent to Ks. Instead, the specific constant (kcat/Km) can besubstituted as a measure of substrate binding; it represents thecatalytic efficiency of the enzyme. The kcat, the catalytic con-stant, represents conversion of the maximum number of reactantmolecules to product per enzyme active site per unit time, or thenumber of turnover events occurring per unit time; it also rep-resents the turnover rate whose unit is the reciprocal of time. Inthe Michaelis-Menten approach, the dissociation of the EP com-plex is slow, and the kcat contribution to this rate constant willbe equal to the dissociation constant. However, in the Briggs-Haldane approach, the dissociation rate of ES complex is fast,and the kcat is equal to k2. In addition, the values of kcat/Km areused not only to compare the efficiencies of different enzymesbut also to compare the efficiencies of different substrates for aspecified enzyme.

Deviations of expected hyperbolic reaction are occasionallyfound due to such factors as experimental artifacts, substrateinhibition, existence of two or more enzymes competing forthe same substrate, and the cooperativity. They show nonhyper-bolic behavior that cannot fit well into the Michaelis-Mentenapproach. For instance, a second molecule binds to the ES com-plex and forms an inactive ternary complex that usually occursat high substrate concentrations and is noticed when rate valuesare lower than expected. It leads to breakdown of the Michaelis-Menten equation. It is not only the Michaelis-Menten plot thatis changed to show a nonhyperbolic behavior; it is also theLineweaver–Burk plot that is altered to reveal the nonlinearityof the curve (see later).

A second example of occasionally occurring nonhyperbolicreactions is the existence of more than one enzyme in a reac-tion that competes for the same substrate; the conditions usuallyare realized when crude or partially purified samples are used.Moreover, the conformational change in the enzymes may beinduced by ligand binding for the regulation of their activities,

as discussed earlier in this chapter; many of these enzymes arecomposed of multimeric subunits and multiple ligand-bindingsites in cases that do not obey the Michaelis-Menten equation.The sigmoid, instead of the hyperbolic, curve is observed; thiscondition, in which the binding of a ligand molecule at one site ofan enzyme may influence the affinities of other sites, is known ascooperativity. The increase and decrease of affinities of enzymebinding sites are the effects of positive and negative cooperativ-ity, respectively. The number of potential substrate binding siteson the enzymes can be quantified by the Hill coefficient, h, andthe degree of cooperativity can be measured by the Hill equation(see page 164). More detailed illustrations and explanations ofdeviations from Michaelis-Menten behavior can be found in theliteratures (Bell and Bell 1988, Cornish-Bowden 1995).

Data Presentation

Untransformed Graphics

The values of Km and Vmax can be determined graphically usingnonlinear plots by measuring the initial rate at various substrateconcentrations and then transforming them into different kineticplots. First, the substrate stock concentration can be chosen at areasonably high level; then, a two-fold (stringently) or five- orten-fold (roughly) serial dilution can be made from this stock.After the data obtained has been evaluated, a Michaelis-Mentenplot of rate vi as a function of substrate concentration [S] issubsequently drawn, and the values of both Km and Vmax canbe estimated. Through the plot, one can check if a hyperboliccurve is observed, and if the values estimated are meaningful.Both values will appear to be infinite if the range of substrateconcentrations is low; on the other hand, although the valueof Vmax can be approximately estimated, that of Km cannot bedetermined if the range of substrate concentrations is too high.Generally, substrate concentrations covering 20–80% of Vmax,which corresponds to a substrate concentration of 0.25–5.0 Km,is appreciated.

Lineweaver–Burk Plots

Though nonlinear plots are useful in determining the values ofKm and Vmax, transformed, linearized plots are valuable in deter-mining the kinetics of multisubstrate enzymes and the interactionbetween enzymes and inhibitors. One of the best known plotsis the double-reciprocal or Lineweaver–Burk plot (Lineweaverand Burk 1934). Inverting both sides of the Michaelis-Mentenequation gives the Lineweaver–Burk plot: 1/v = (Km/Vmax)(1/[S]) + 1/Vmax (Fig. 8.3). The equation is for a linear curvewhen one plots 1/v against 1/[S] with a slope of Km/Vmax and ay intercept of 1/Vmax. Thus, the kinetic values can also be deter-mined from the slope and intercept values of the plot. However,caution should be taken due to small experimental errors thatmay be amplified by taking the reciprocal (the distorting effect),especially in the measurement of v values at low substrate con-centrations. The problem can be solved by preparing a series ofsubstrate concentrations evenly spaced along the 1/[S] axis, that

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Figure 8.3. The Lineweaver–Burk double-reciprocal plot.

is, diluting the stock solution of substrate by two-, three-, four-,five-, . . . fold (Copeland 2000).

Eadie–Hofstee Plots

The Michaelis-Menten equation can be rearranged to givev = −Km (v/[S]) + Vmax by first multiplying both sides byKm + [S], and then dividing both sides by [S]. The plot will givea linear curve with a slope of −Km and a y intercept of v whenv is plotted against v/[S] (Fig. 8.4). The Eadie–Hofstee plot hasthe advantage of decompressing data at high substrate concen-trations and being more accurate than the Lineweaver–Burk plot,but the values of v against [S] are more difficult to determine(Eadie 1942, Hofstee 1959). It also has the advantage of observ-ing the range of v from zero to Vmax and is the most effectiveplot for revealing deviations from the hyperbolic reaction whentwo or more components, each of which follows the Michaelis-Menten equation, although errors in v affect both coordinates(Cornish-Bowden 1996).

Figure 8.4. The Eadie–Hofstee plot.

Figure 8.5. The Hanes–Wolff plot.

Hanes–Wolff Plots

The Lineweaver–Burk equation can be rearranged by multiply-ing both sides by [S]. This gives a linear plot [S]/v = (1/Vmax)[S] + Km/Vmax, with a slope of 1/Vmax, the y intercept ofKm/Vmax, and the x intercept of −Km when [S]/v is plottedas a function of [S] (Fig. 8.5). The plot is useful in obtainingkinetic values without distorting the data (Hanes 1932).

Eisenthal–Cornish-Bowden Plots(the Direct Linear Plots)

As in the Michaelis-Menten plot, pairs of rate values of v andthe negative substrate concentrations [S] are applied along they and x axis, respectively. Each pair of values is a linear curvethat connects two points of values and extrapolates to pass theintersection. The location (x, y) of the point of intersection onthe two-dimensional plot represents Km and Vmax, respectively(Fig. 8.6). The direct linear plots (Eisenthal and Cornish-Bowden

Figure 8.6. The Eisenthal–Cornish-Bowden direct plot.

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Figure 8.7. The Hill plot.

1974) are useful in estimating the Michaelis-Menten kineticparameters and are the better plots for revealing deviations fromthe reaction behavior.

Hill Plot

The Hill equation is a quantitative analysis measuringnon–Michaelis-Menten behavior of cooperativity (Hill 1910).In a case of completely cooperative binding, an enzyme con-tains h binding sites that are all occupied simultaneously witha dissociation constant K = [E][S]h/[ESh], where h is the Hillcoefficient, which measures the degree of cooperativity. If thereis no cooperativity, h = 1; there is positive cooperativity whenh > 1, negative cooperativity when h < 1. The rate value canbe expressed as v = Vmax[S]h/(K + [S]h), and the Hill equa-tion gives a linear log(vi/Vmax − v) = h log[S] − log K , whenlog(v/Vmax − v) is plotted as a function of log[S] with a slope ofh and a y intercept of − log K (Fig. 8.7). However, the equationwill show deviations from linearity when outside a limited rangeof substrate concentrations, that is, in the range of [S] = K.

FACTORS AFFECTING ENZYMEACTIVITYAs discussed earlier in this chapter, the rate of an enzymatic reac-tion is very sensitive to reaction conditions such as temperature,pH, ionic strength, buffer constitution, and substrate concentra-tion. To investigate the catalytic mechanism and the efficiencyof an enzyme with changes in the parameters, and to evaluate asuitable circumstance for assaying the enzyme activity, the con-ditions should be kept constant to allow reproducibility of dataand to prevent a misleading interpretation.

Enzyme, Substrate, and CofactorConcentrations

In general, the substrate concentration should be kept muchhigher than that of enzyme in order to prevent substrate concen-

tration dependency of the reaction rate at low substrate concen-trations. The rate of an enzyme-catalyzed reaction will also showlinearity to the enzyme concentration when substrate concentra-tion is constant. The reaction rate will not reveal the linearityuntil the substrate is depleted, and the measurement in initial ratewill be invalid. Preliminary experiments must be performed todetermine the appropriate range of substrate concentrations overa number of enzyme concentrations to prevent the phenomenonof substrate depletion. In addition, the presence of inhibitor, acti-vator, and cofactor in the reaction mixture also influence enzymeactivity. The enzymatic activity will be low or undetectable inthe presence of inhibitors or in the absence of activators or co-factors. In the case of cofactors, the activated enzymes will beproportional to the cofactor concentration added in the reactionmixture if enzymes are in excess. The rate of reaction will notrepresent the total amount of enzyme when the cofactor sup-plement is not sufficient, and the situation can be avoided byadding excess cofactors (Tipton 1992). Besides, loss of enzymeactivity may be seen before substrate depletion, as when theenzyme concentration is too low and leads to the dissociationof the dimeric or multimeric enzyme. To test this possibility,addition of the same amount of enzyme can be applied to thereaction mixture to measure if there is the appearance of anotherreaction progression curve.

Effects of pH

Enzymes will exhibit maximal activity within a narrow range ofpH and vary over a relatively broad range. For an assay of en-zymatic catalysis, the pH of the solution of the reaction mixturemust be maintained in an optimal condition to avoid pH-inducedprotein conformational changes, which lead to diminishment orloss of enzyme activity. A buffered solution, in which the pH isadjusted with a component with pKa at or near the desired pHof the reaction mixture, is a stable environment that provides theenzyme with maximal catalytic efficiency. Thus, the appropriatepH range of an enzyme must be determined in advance whenoptimizing assay conditions.

To determine if the enzymatic reaction is pH dependent and tostudy the effects of group ionizations on enzyme kinetics, the rateas a function of substrate concentration at different pH conditionscan be measured to simultaneously obtain the effects of pH on thekinetic parameters. It is known that the ionizations of groups areof importance, either for the active sites of the enzyme involvedin the catalysis or for maintaining the active conformation ofthe enzyme. Plots of kcat, Km, and kcat/Km values at varyingpH ranges will reflect important information about the roles ofgroups of the enzyme. The effect of pH dependence on the valueof kcat reveals the steps of ionization of groups involved in theES complex and provides a pKa value for the ES complex state;the value of Km shows the ionizing groups that are essentialto the substrate-binding step before the reaction. Moreover, thekcat/Km value reveals the ionizing groups that are essential toboth the substrate-binding and the ES complex–forming steps,and provides the pKa value of the free reactant molecules state(Brocklehurst and Dixon 1977, Copeland 2000). From a plot ofthe kcat/Km value on the logarithmic scale as a function of pH,

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Figure 8.8. The effect of pH on the kcat/Km value of an enzymaticreaction that is not associated with the Henderson-Hasselbalchequation.

the pKa value can be determined from the point of intersectionof lines on the plot, especially for an enzymatic reaction that isnot associated with the Henderson-Hasselbalch equation pH =pKa + log ([A−]/[HA]) (Fig. 8.8). Thus, the number of ionizinggroups involved in the reaction can be evaluated (Dixon andWebb 1979, Tipton and Webb 1979).

Effects of Temperature

Temperature is one of the important factors affecting enzymeactivity. For a reaction to occur at room temperature without thepresence of an enzyme, small proportions of reactant moleculesmust have sufficient energy levels to participate in the reaction(Fig. 8.9A). When the temperature is raised above room tem-perature, more reactant molecules gain enough energy to beinvolved in the reaction (Fig. 8.9B). The EA is not changed,but the distribution of energy-sufficient reactants is shifted to ahigher average energy level. When an enzyme is participating inthe reaction, the EA is lowered significantly, and the proportionof reactant molecules at an energy level above the activationenergy is also greatly increased (Fig. 8.9C). That means thereaction will proceed at a much higher rate.

Most enzyme-catalyzed reactions are characterized by an in-crease in the rate of reaction and increased thermal instabilityof the enzyme with increasing temperature. Above the criticaltemperature, the activity of the enzyme will be reduced signifi-cantly; while within this critical temperature range, the enzymeactivity will remain at a relatively high level, and inactivation ofthe enzyme will not occur. Since the rate of reaction increasesdue to the increased temperature by lowering the activation en-ergy EA, the relationship can be expressed by the Arrheniusequation: k = A exp(−EA/RT ), where A is a constant relatedto collision probability of reactant molecules, R is the ideal gasconstant (1.987 cal/mol—deg), T is the temperature in degreesKelvin (K = ◦C + 273.15), and k represents the specific rateconstant for any rate, that is, kcat or Vmax. The equation can be

Figure 8.9. Plots of temperature effect on the energy levels ofreactant molecules involved in a reaction. (A) The first plot depictsthe distribution of energy levels of the reactant molecules at roomtemperature without the presence of an enzyme. (B) The secondplot depicts that at the temperature higher than room temperaturebut in the absence of an enzyme. (C) The third plot depicts that atroom temperature in the presence of an enzyme. The vertical line ineach plot indicates the required activation energy level for a reactionto occur. The shaded portion of distribution in each plot indicates theproportion of reactant molecules that have enough energy levels tobe involved in the reaction. The x-axis represents the energy level ofreactant molecules, while the y-axis represents the frequency ofreactant molecules at an energy level.

transformed into: ln K = ln A − EA/RT , where the plot of ln K

against 1/T usually shows a linear relationship with a slope of−EA/R, where the unit of EA is cal/mol. The calculated EA

of a reaction at a particular temperature is useful in predictingthe EA of the reaction at another temperature. And the plot is

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useful in judging if there is a change in the rate-limiting stepof a sequential reaction when the line of the plot revealsbending of different slopes at certain temperatures (Stauffer1989).

Taken together, the reaction should be performed under aconstantly stable circumstance, with both temperature and pHprecisely controlled from the start to the finish of the assay,for the enzyme to exhibit highly specific activity at appropriateacidbase conditions and buffer constitutions. Whenever possi-ble, the reactant molecules should be equilibrated at the requiredassay condition following addition of the required componentsand efficient mixing to provide a homogenous reaction mixture.Because the enzyme to be added is usually stored at lowtemperature, the reaction temperature will not be significantlyinfluenced when the enzyme volume added is at as low as 1–5%of the total volume of the reaction mixture. A lag phase will benoticed in the rate measurement as a function of time when thetemperature of the added enzyme stock eventually influencesthe reaction. Significant temperature changes in the componentsstored in different environments and atmospheres should beavoided when the reaction mixture is mixed and the assay hasstarted.

METHODS USED IN ENZYME ASSAYSGeneral Considerations

A suitable assay method is not only a prerequisite for detectingthe presence of enzyme activity in the extract or the purified ma-terial: it is also an essential vehicle for kinetic study and inhibitorevaluation. Selection of an assay method that is appropriate tothe type of investigation and the purity of the assaying materialis of particular importance. It is known that the concentrationof the substrate will decrease and that of product will increasewhen the enzyme is incubated with its substrate. Therefore,an enzyme assay is intended to measure either the decrease insubstrate concentration or the increase in product concentrationas a function of time. Usually, the latter is preferred becausea significant increase of signal is much easier to monitor. It isrecommended that a preliminary test be performed to determinethe optimal conditions for a reaction including substrateconcentration, reaction temperature, cofactor requirements, andbuffer constitutions, such as pH and ionic strength, to ensurethe consistency of the reaction. Also, an enzyme blank shouldbe performed to determine if the nonenzymatic reaction isnegligible or could be corrected. When a crude extract, ratherthan purified enzyme, is used for determining enzyme activity,one control experiment should be performed without addingsubstrate to determine if there are any endogenous substratespresent and to prevent overcounting of the enzyme activity inthe extract.

Usually, the initial reaction rate is measured, and it is re-lated to the substrate concentration. Highly sufficient substrateconcentration is required to prevent a decrease in concentrationduring the assay period of enzyme activity, thus allowing theconcentration of the substrate to be regarded as constant.

Types of Assay Methods

On the basis of measuring the decrease of the substrate or theincrease of the product, one common characteristic propertythat is useful for distinguishing the substrate and the product istheir absorbance spectra, which can be determined using one ofthe spectrophotometric methods. Observation of the change inabsorbance in the visible or near UV spectrum is the methodmost commonly used in assaying enzyme activity. NAD(P)+

is quite often a cofactor of dehydrogenases, and NAD(P)H isthe resulting product. The absorbance spectrum is 340 nm forthe product, NAD(P)H, but not for the cofactor, NAD (P)+. Acontinuous measurement at absorbance 340 nm is required tocontinuously monitor the disappearance of the substrate or theappearance of the product. This is called a continuous assay,and it is also a direct assay because the catalyzed reaction it-self produces a measurable signal. The advantage of continuousassays is that the progress curve is immediately available, asis confirmation that the initial rate is linear for the measuringperiod. They are generally preferred because they give more in-formation during the measurement. However, methods based onfluorescence, the spectrofluorometric methods, are more sensi-tive than those based on the changes in the absorbance spectrum.The reaction is accomplished by the release or uptake of protonsand is the basis of performing the assay. Detailed discussions onthe assaying techniques are available below.

Sometimes, when a serial enzymatic reaction of a metabolicpathway is being assayed, no significant products, the substratesof the next reaction, can be measured in the absorbance spectradue to rapid changes in the reaction. Therefore, there may be nosuitable detection machinery available for this single enzymaticstudy. Nevertheless, there is still a measurable signal when oneor several enzymatic reactions are coupled to the desired assayreaction. These are coupled assays, one of the particular indirectassays whose coupled reaction is not enzymatic. This meansthat the coupled enzymes and the substrates have to be presentin excess to make sure that the rate-limiting step is always thereaction of the particular enzyme being assayed. Though a lagin the appearance of the preferred product will be noticed atthe beginning of the assay, before reaching a steady state, thephenomenon will only appear for a short period of time if theconcentration of the coupling enzymes and substrates are keptin excess. So, the Vmax of the coupling reactions will be greaterthan that of the preferred reaction. Besides, not only the substrateconcentration but also other parameters may affect the preferredreaction (see previously). To prevent this, control experimentscan be performed to check if the Vmax of the coupling reactionsis actually much higher than that of the preferred reaction. Forother indirect assays in which the desired catalyzed reactionitself does not produce a measurable signal, a suitable reagentthat has no effect on enzyme activity can be added to the reactionmixture to react with one of the products to form a measurablesignal.

Nevertheless, sometimes no easily readable differences be-tween the spectrum of absorbance of the substrate and that ofthe product can be measured. In such cases, it may be possi-ble to measure the appearance of the colored product, by the

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chromogenic method (one of the spectrofluorometric methods).The reaction is incubated for a fixed period of time. Then, be-cause the development of color requires the inhibition of enzymeactivity, the reaction is stopped and the concentration of coloredproduct of the substrate is measured. This is the discontinuousor end point assay. Assays involving product separation thatcannot be designed to continuously measure the signal change,such as electrophoresis, high performance liquid chromatogra-phy (HPLC), and sample quenching at time intervals, are alsodiscontinuous assays. The advantage of discontinuous assays isthat they are less time consuming for monitoring a large num-ber of samples for enzyme activity. However, additional controlexperiments are needed to ensure that the initiation rate is linearfor the measuring period of time. Otherwise, the radiolabeledsubstrate of an enzyme assay is a highly sensitive method thatallows the detection of radioactive product. Separation of thesubstrate and the product by a variety of extraction methodsmay be required to accurately assay the enzyme activity.

Detection Methods

Spectrophotometric Methods

Both the spectrophotometric method and the spectrofluoromet-ric method discussed below use measurements at specific wave-lengths of light energy (in the wavelength regions of 200–400 nm(UV, ultraviolet) and 400–800 nm (visible)) to determine howmuch light has been absorbed by a target molecule (resulting inchanges in electronic configuration). The measured value fromthe spectrophotometric method at a specific wavelength can berelated to the molecule concentration in the solution using a cellwith a fixed path length (l cm) that obeys the Beer-Lambert law:

A = − log T = εcl,

where A is the absorbance at certain wavelength, T is the trans-mittance, representing the intensity of transmitted light, ε is theextinction coefficient, and c is the molar concentration of thesample. Using this law, the measured change in absorbance,�A, can be converted to the change in molecule concentration,�c, and the change in rate, vi, can be calculated as a function oftime, �t , that is, vi = �A/εl�t .

Choices of appropriate materials for spectroscopic cells (cu-vettes) depend on the wavelength used; quartz cuvettes must beused at wavelengths less than 350 nm because glass and dis-posable plastic cuvettes absorb too much light in the UV lightrange. However, the latter glass and disposible plastic cuvettescan be used in the wavelength range of 350–800 nm. Selectionof a wavelength for the measurement depends on finding thewavelength that produces the greatest difference in absorbancebetween the reactant and product molecules in the reaction.Though the wavelength of measurement usually refers to themaximal wavelength of the reactant or product molecule, themost meaningful analytical wavelength may not be the same asthe maximum wavelength because significant overlap of spec-tra may be found between the reactant and product molecules.Thus, a different spectrum between two molecules can be calcu-

lated to determine the most sensitive analytical wavelengths formonitoring the increase in product and the decrease in substrate.

Spectrofluorometric Methods

When a molecule absorbs light at an appropriate wavelength,an electronic transition occurs from the ground state to the ex-cited state; this short-lived transition decays through varioushigh-energy, vibrational substrates at the excited electronic stateby heat dissipation, and then relaxes to the ground state with aphoton emission, the fluorescence. The emitted fluorescence isless energetic (longer wavelength) than the initial energy that isrequired to excite the molecules; this is referred to as the Stokesshift. Taking advantage of this, the fluorescence instrument isdesigned to excite the sample and detect the emitted light at dif-ferent wavelengths. The ratio of quanta fluoresced over quantaabsorbed offers a value of quantum yield (Q), which measuresthe efficiency of the reaction leading to light emission. The lightemission signals vary with concentration of fluorescent mate-rial by the Beer-Lambert law, where the extinction coefficientis replaced by the quantum yield, and gives: If = 2.31 I0εclQ,where If and I0 are the intensities of light emission and of in-cident light that excites the sample, respectively. However, con-version of light emission values into concentration units requiresthe preparation of a standard curve of light emission signals asa function of the fluorescent material concentration, which hasto be determined independently, due to the not strictly linearrelationship between If and c (Lakowicz 1983).

Spectroflurometric methods provide highly sensitive capac-ities for detection of low concentration changes in a reaction.However, quenching (diminishing) or resonance energy transfer(RET) of the measured intensity of the donor molecule willbe observed if the acceptor molecule absorbs light and thedonor molecule emits light at the same wavelength. The acceptormolecule will either decay to its ground state if quenching occursor fluoresce at a characteristic wavelength if energy is trans-ferred (Lakowicz 1983). Both situations can be overcome byusing an appropriate peptide sequence, up to 10 or more aminoacid residues, to separate the fluorescence donor molecule fromthe acceptor molecule if peptide substrates are used. Thus, botheffects can be relieved by cleaving the intermolecular bondingwhen protease activity is being assayed.

Care should be taken in performing the spectrofluorometricmethod. For instance, the construction of a calibration curveas the experiment is assayed is essential, and the storage con-ditions for the fluorescent molecules are important because ofphotodecomposition. In addition, the quantum yield is related tothe temperature, and the fluorescence signal will increase withdecreasing temperature, so a temperature-constant condition isrequired (Bashford and Harris 1987, Gul et al. 1998).

Radiometric Methods

The principle of the radiometric methods in enzymatic catal-ysis studies is the quantification of radioisotopes incorporatedinto the substrate and retained in the product. Hence, success-ful radiometric methods rely on the efficient separation of the

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radiolabled product from the residual radiolabeled substrate andon the sensitivity and specificity of the radioactivity detectionmethod. Most commonly used radioisotopes, for example, 14C,32P, 35S, and 3H, decay through emission of β particles; how-ever, 125I decays through emission of γ particles, whose loss isrelated to loss of radioactivity and the rate of decay (the half-life)of the isotope. Radioactivity expressed in Curies (Ci) decays at arate of 2.22 × 1012 disintegrations per minute (dpm); expressedin Becquerels (Bq), it decays at a rate of 1 disintegrations persecond (dps). The experimental units of radioactivity are countsper minute (cpm) measured by the instrument; quantification ofthe specific activity of the sample is given in units of radioac-tivity per mass or per molarity of the sample (e.g., µCi/mg ordpm/µmol).

Methods of separation of radiolabeled product and residualradiolabeled substrate include chromatography, electrophoresis,centrifugation, and solvent extraction (Gul et al. 1998). For thedetection of radioactivity, one commonly used instrument is ascintillation counter that measures light emitted when solutionsof p-terphenyl or stilbene in xylene or toluene are mixed withradioactive material designed around a photomultiplier tube.Another method is the autoradiography that allows detecting ra-dioactivity on surfaces in close contact either with X-ray film orwith plates of computerized phosphor imaging devices. When-ever operating the isotopescontaining experiments, care shouldalways be taken for assuring safety.

Chromatographic Methods

Chromatography is applied in the separation of the reactantmolecules and products in enzymatic reactions and is usuallyused in conjunction with other detection methods. The mostcommonly used chromatographic methods include paper chro-matography, column chromatography, thin-layer chromatogra-phy (TLC), and HPLC. Paper chromatography is a simple andeconomic method for the readily separation of large numbersof samples. By contrast, column chromatography is more ex-pensive and has poor reproducibility. The TLC method has theadvantage of faster separation of mixed samples, and like pa-per chromatography, it is disposable and can be quantified andscanned; it is not easily replaced by HPLC, especially for mea-suring small, radiolabeled molecules (Oldham 1992).

The HPLC method featured with low compressibility resinsis a versatile method for the separation of either low molecularweight molecules or small peptides. Under a range of high pres-sures up to 5000 psi (which approximates to 3.45 × 107 Pa),the resolution is greatly enhanced with a faster flow rate anda shorter run time. The solvent used for elution, referred to asthe mobile phase, should be an HPLC grade that contains lowcontaminants; the insoluble media is usually referred to as thestationary phase. Two types of mobile phase are used duringelution; one is an isocratic elution whose composition is notchanged, and the other is a gradient elution whose concentra-tion is gradually increased for better resolution. The three HPLCmethods most commonly used in the separation steps of enzy-matic assays are reverse phase, ion-exchange, and size-exclusionchromatography.

The basis of reverse phase HPLC is the use of a nonpolarstationary phase composed of silica covalently bonded to alkylsilane, and a polar mobile phase used to maximize hydrophobicinteractions with the stationary phase. Molecules are eluted in asolvent of low polarity (e.g., methanol, acetonitrile, or acetonemixed with water at different ratios) that is able to efficientlycompete with molecules for the hydrophobic stationary phase.

The ion-exchange HPLC contains a stationary phase co-valently bonded to a charged functional group; it binds themolecules through electrostatic interactions, which can be dis-rupted by the increasing ionic strength of the mobile phase.By modifying the composition of the mobile phase, differentialelution, separating multiple molecules, is achieved.

In the size-exclusion HPLC, also known as gel filtration, thestationary phase is composed of porous beads with a particularmolecular weight range of fractionation. However, this method isnot recommended where molecular weight differences betweensubstrates and products are minor, because of overlapping of theelution profiles (Oliver 1989).

Selection of the HPLC detector depends on the types of signalsmeasured, and most commonly the UV/visible light detectors areextensively used.

Electrophoretic Methods

Agarose gel electrophoresis and polyacrylamide gel elec-trophoresis (PAGE) are widely used methods for separation ofmacromolecules; they depend, respectively, on the percentageof agarose and acrylamide in the gel matrix. The most com-monly used method is the sodium dodecyl sulfate (SDS)-PAGEmethod; under denaturing conditions, the anionic detergentSDS is coated on peptides or proteins giving them equivalentlythe same anionic charge densities. Resolving of the samples willthus be based on molecular weight under an electric field over aperiod of time. After electrophoresis, peptides or proteins bandscan be visualized by staining the gel with Coomassie BrilliantBlue or other staining reagents, and radiolabeled materials canbe detected by autoradiography. Applications of electrophoresisassays are not only for detection of molecular weight andradioactivity differences, but also for detection of chargedifferences. For instance, the enzyme-catalyzed phosphoryla-tion reactions result in phosphoryl transfer from substrates toproducts, and net charge differences between two moleculesform the basis for separation by electrophoresis. If radioisotope32P-labeled phosphate is incorporated into the molecules, thereactions can be detected by autoradiography, by monitoringthe radiolabel transfer after gel electrophoresis, or by immuno-logical blotting with antibodies that specifically recognizepeptides or proteins containing phosphate-modified amino acidresidues.

Native gel electrophoresis is also useful in the above ap-plications, where not only the molecular weight but also thecharge density and overall molecule shape affect the migrationof molecules in gels. Though SDS-PAGE causes denaturing topeptides and proteins, renaturation in gels is possible and canbe applied to several types of in situ enzymatic activity studiessuch as activity staining and zymography (Hames and Rickwood

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1990). Both methods assay enzyme activity after electrophore-sis, but zymography is especially intended for proteolytic en-zyme activity staining in which gels are cast with high concen-trations of proteolytic enzyme substrates, for example, casein,gelatin, bovine serum albumin, collagen, and others. Samplescontaining proteolytic enzymes can be subjected to gel elec-trophoresis, but the renaturation step has to be performed if adenaturing condition is used; then the reaction is performed un-der conditions suitable for assaying proteolytic enzymes. Thegel is then subjected to staining and destaining, but the entire gelbackground will not be destained because the gel is polymerizedwith protein substrates, except in clear zones where the signifi-cant proteolysis has occurred; the amount of staining observedwill be greatly diminished due to the loss of protein. This processis also known as reverse staining. Otherwise, reverse zymogra-phy is a method used to assay the proteolytic enzyme inhibitoractivity in gel. Similar to zymography, samples containing prote-olytic enzyme inhibitor can be subjected to gel electrophoresis.After the gel renaturation step is performed, the reaction is as-sayed under appropriate conditions in the presence of a specifictype of proteolytic enzyme. Only a specific type of proteolyticenzyme inhibitor will be resistant to the proteolysis, and afterstaining, the active proteolytic enzyme inhibitor will appear asprotein band (Oliver et al. 1999).

Other Methods

The most commonly used assay methods are the spectrophoto-metric, spectrofluorometric, radiometric, chromatographic, andelectrophoretic methods described above, but a variety of othermethods are utilized as well. Immunological methods make useof the antibodies raised against the proteins (Harlow and Lane1988). Polarographic methods make use of the change in cur-rent related to the change in concentration of an electroactivecompound that undergoes oxidation or reduction (Vassos andEwing 1983). Oxygen-sensing methods make use of the changein oxygen concentration monitored by an oxygen-specific elec-trode (Clark 1992), and pH-stat methods use measurements ofthe quantity of base or acid required to be added to maintain aconstant pH (Jacobsen et al. 1957).

Selection of an Appropriate Substrate

Generally, a low molecular mass, chromogenic substrate con-taining one susceptible bond is preferred for use in enzymaticreactions. A substrate containing many susceptible bonds or dif-ferent functional groups adjacent to the susceptible bond mayaffect the cleavage efficiency of the enzyme, and this can re-sult in the appearance of several intermediate-sized products.Thus, they may interfere with the result and make interpretationof kinetic data difficult. Otherwise, the chromophore-containingsubstrate will readily and easily support assaying methods withabsorbance measurement. Moreover, substrate specificity canalso be precisely determined when an enzyme has more than onerecognition site on both the preferred bond and the functionalgroups adjacent to it. Different sized chromogenic substrates

can then be used to determine an enzyme’s specificity and toquantify its substrate preference.

Unit of Enzyme Activity

The unit of enzyme activity is usually expressed as either micro-moles of substrate converted or product formed per unit time,or unit of activity per milliliter under a standardized set of con-ditions. Though any unit of enzyme activity can be used, theCommission on Enzymes of the International Union of Bio-chemistry and Molecular Biology (IUBMB) has recommendedthat a unit of enzyme, Enzyme Unit or International Unit (U),be used. An Enzyme Unit is defined as that amount that willcatalyze the conversion of 1 micromole of substrate per minute,1 U = 1 µmol/min, under defined conditions. The conditionsinclude substrate concentration, pH, temperature, buffer compo-sition, and other parameters that may affect the sensitivity andspecificity of the reaction, and usually a continuous spectropoto-metric method or a pH stat method is preferred. Another enzymeunit that now is not widely used is the International System ofUnits (SI unit) in which 1 katal (kat) = 1 mol/sec, so 1 kat =60 mol/min = 6 × 107 U.

In ascertaining successful purification of a specified enzymefrom an extract, it is necessary to compare the specific activityof each step to that of the original extract; a ratio of the twogives the fold purification. The specific activity of an enzymeis usually expressed as units per milligram of protein when theunit of enzyme per milliliter is divided by milligrams of proteinper milliliter, the protein concentration. The fold purification isan index reflecting only the increase in the specific activity withrespect to the extract, not the purity of the specified enzyme.

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Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Enzyme Activities