FHSU BIOCHEMISTRY LABORATORY SERIES - Fall 2010

NAM E Lab 521

ENZYMES: KINETICS OF INVERTASE

SECTION LOCKER

INSTRUCTOR

DATE

INTRODUCTION Am ong the various functions of proteins, the m ost dram atic, perhaps, is that of enzym e activity. Enzym es m ediate, control, and catalyze the chem ical affairs of a living cell. Although the action of enzym es has been studied for over 100 years, the m anner in which an enzym e protein m olecule functions is still not com pletely understood. Practically speaking, enzym e technology has existed for centuries, as in wine m aking. In fact, m uch of our early understanding of enzym e action resulted from the work of Pasteur, Kuhne, Bchner, and others investigating the action of yeast. In his studies, Kuhne, in 1878, first introduced the term enzym e (Greek "in yeast"). Enzym es are a com m on part of our practical daily lives; for exam ple, you can buy enzym es to tenderize m eat. Cheese was and still is m ade by treating m ilk with an enzym e called rennin, obtained from the lining of a calf's stom ach. Ferm entation has always been an im portant art. The protein nature of enzym es was not dem onstrated conclusively until 1926, when Sum ner prepared crystals of the enzym e urease and showed them to be protein. Since then over 150 enzym es have been crystallized and over 1000 have been isolated in varying degrees of purity. This is not very m any when you consider that even a sim ple organism such as E. coli has about 3000 different proteins. In cells of higher anim als, the num ber of different proteins m ay range in the m illions. In m any ways, the study of enzym es has provided a bridge between the biological and physical sciences. Investigations of the structure, action, and properties of enzym es will occupy chem ists, biologists, and physicists for m any years to com e. Am ong the earliest known and m ost widely studied of enzym es are those that hydrolyze sucrose. Berthelot, in 1860, working on the purification of a sucrose hydrolyzing enzym e from yeast, nam ed the enzym e ferm ent inversif because of the inversion (+ to -) in the optical rotation during the hydrolysis of sucrose. Subsequently, the nam es invertase, sucrase, saccharase, and m ore recently, &fructofuranosidase have been used with reference to the enzym atic hydrolysis of sucrose. Invertase activity has been dem onstrated in yeasts, m olds, m any bacteria, plants, and higher anim als. The basic reaction for yeast invertase is that of a &fructofuranosidase. Many &-fructofuranosides are hydrolyzed by the enzym e but the activity of the

enzym e towards sucrose far exceeds that towards other natural substrates; for exam ple, raffinose or stachyose. The hydrolytic reaction is m ost conveniently assayed by observing the change in optical rotation (a kinetic assay) or in reducing power (a fixed tim e assay) upon incubation of the enzym e with sucrose. This experim ent uses the fixed tim e assay procedure, which consists of stopping the reaction with alkali and subsequently m easuring the reducing power by m eans of the 3,5-dinitrosalicylate (DNS) m ethod. QUANTITATIVE DETERM INATION OF GLUCOSE Num erous m ethods of quantitative determ ination of sugars have been used. The m ost com m only used m ethods all rely on the ability of sugars to reduce various substances. This seriously lim its the sugars that can be determ ined since several sugars are not oxidized. However, for the m onosaccharides and som e disaccarides this is the easiest, quickest, and the m ost reliable m ethod. Several m ethods which rely on the reducing power of sugars are Nelsons Reagent, Benedicts Reagent, and dinitrosalicylic acid. In this laboratory experim ent dinitrosalicylic acid (DNS) reagent will be used. In this m ethod 3,5dinitrosalicylic acid is reduced in alkaline solution to from an intensely colored, orange product which is m ost likely 3-am ino-5-nitrosalicylic acid. The product of the reaction absorbs at 540 nm whereas the starting m aterial absorbs only sightly at this wavelength. The change in absorption at 540 nm has been shown to be proportional to the am ount of sugar present. Experimental Procedure Prepare a glucose standard curve by following the protocol. Tube No. 1 2 3 4 5 6 mL of 4x10 -3 M Glc 0.0 0.2 0.4 0.6 0.8 1.0 mL H 2O 1.0 0.8 0.6 0.4 0.2 0.0 mL DNS Reagent 1.0 1.0 1.0 1.0 1.0 1.0

W hen all additions have been m ade, thoroughly m ix the contents and heat the tubes in a boiling water bath for 5 m inutes. Rem ove the tubes and cool to room tem perature in running tap water for 2-3

521 - ENZYM ES - Page 2 m inutes and add 5 m L of distilled water. Measure the absorbance of each solution at 540 nm using tube num ber one as a blank. Reference J.B. Sum ner, J. Biol. Chem . 65, 393 (1925) DETERM INATION OF ENZYM E CONCENTRATION AND SPECIFIC ACTIVITY Enzym e concentration is norm ally determ ined by either the am ount of product form ed or the am ount of substrate transform ed. Under a certain set of conditions a plot of velocity versus enzym e concentration should yield a linear relationship. The in i tia l st ep in w or k wi th a ny enzym e should be to find these conditions. Experimental Procedure Prepare a dilution of enzym e (usually either 1:500 or 1:600) using gelatin as the diluent. Determ ine the enzym e concentration by using the following protocol. 1.0 m L aliquot of the assay m ixture and adding it to a tube which contains 1.0 m L of DNS reagent. Develop the color in all tubes--assays and blank--as previously done for preparation of the DNS standard curve. The blank in this case should be tube num ber 1. The specific activity of an enzym e is defined in term s of units/m g. of protein. Expression of enzym e concentration in these term s is used prim arily during purification of the enzym e. In such a case an increase in specific activity indicates an increase in purity. Dilute a sm all portion of the stock e n z y m e solution with w a t e r (usually 1:50 is adequate). Use the following protocol to determ ine the protein concentration by the Folin m ethod. Review the exact procedure for the Folin m ethod (Exp. 503) before proceeding. Calculations The data collected in the experim ent will be in term s of absorbance at 540 nm . This should be converted to velocity which is m oles of sucrose hydrolyzed per m inute. To accom plish the conversion use the DNS standard curve and determ ine the num ber of m ol es of g lu c o s e that is e q u iv alent to the absorbance. The m oles of glucose tim es 0.3 yields velocity. The value of 0.3 is used because (a) the equivalent of 2 glucoses are produced from 1 sucrose, (b) 1 m L is analyzed from the total volum e of 3 m L, and (c) the reaction is run for 5 m inutes.

Add the enzym e, water, and buffer (0.05M acetate buffer, pH 4.7) allow the tubes to equilibrate at 30EC for at least two m inutes. Initiate the reaction by adding 1 m L of 0.3M sucrose which has also been equilibrated at 30EC for two m inutes. Incubate each assay m ixture for exactly five m inutes. Start and stop the individual assays at 30 or 60 second intervals to insure assays of exactly 5

W hen velocity is plotted versus m L of enzym e, the slope of the line will give the num ber of m oles of sucrose hydrolyzed per m inute per m L of enzym e. Since a unit of enzym e concentration is defined as the am ount of enzym e that hydrolyzes 1 m ole of sucrose in 1 m inute, the slope of the line also yields the enzym e concentration in units per m L. Multiplying this by the dilution factor gives the concentration of the stock enzym e solution. To calculate the specific activity the protein concentration m ust also be known.

From the Folin determ ination, determ ine the num ber of m g/m L, m ultiply by the dilution factor, and calculate the specific activity. m inute duration. Stop the reaction by rem oving a pH OPTIM UM OF ENZYM ATIC HYDROLYSIS

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Prepare a dilution of enzym e which yields a high rate of hydrolysis when assayed under the standard condition. Determ ine the pH optim um for invertase by the following protocol. Prepare the six tubes by adding the citrate-EDTA buffer, 0.3M sucrose, and water. After two m inutes initiate the reaction by the addition of 1.0 m L of enzym e. After exactly five m inutes stop the reaction as previously described. Develop the color

enzyme the same was used in the Km - Vm a x determination.

Com bine the 0.05M acetate buffer (pH 4.7), enzym e, 1.5 x 10 -4M p-hydroxym ercuribenzoate (PMB), and water. Incubate for two m inutes at 30EC and incubate the reaction by adding 1.0 m L of sucrose. After 5 m inutes stop the reaction and analyze the results in the usual m anner. If the results are not satisfactory repeat this portion of the experim ent using 0.2 m L of 7.5 x10 -5M PMB (prepared by m ixing equal volum es of 1.5 x10 -4M PMB and water). Calculations

and determ ine the absorbance using tube num ber 1 as a blank. Change the absorbance readings into velocity and record these values. DETERM INATION OF K m, V max AND INHIBITION The tw o portions of this experiment (w ith and w ithout inhibitor) must be conducted on the same day with the same dilution of enzyme. Prepare a dilution of enzym e that gives a high rate of hydrolysis when assayed under standard conditions. You should have at least 15 m L of the diluted enzym e. Use the following protocol to collect data necessary for determ ining K m and V max.
*These amounts can be varied to obtain an amount of enzyme that will give a high rate of hydrolysis but the total

The calculations require that the substrate concentrations and velocity be used in several ways. The substrate concentration is one third of the concentration that is added because 1.0 m illiliter is added to a m ixture which has a total volum e of 3.0 m illiliters. Likewise the concentration of inhibitor is 1/15th the concentration which is added. EFFECT OF TEM PERATURE ON THE ENZYM E AND ACID CATALYZED HYDROLYSIS OF SUCROSE Prepare an enzym e dilution that is known to yield a velocity that is about 40% of the m axim um . Use the following protocol to determ ine the effect of tem perature on enzym atic reaction. * These values can be varied as necessary to obtain the
proper velocity but the total of the two must be 1.5 mL.

must be 1.5 mL and the amount of enzyme cannot exceed 1.3 mL. #These concentrations are readily prepared by diluting 0.3M sucrose. Dilute enough for this portion of the experiment and the inhibition studies. In the assay tubes place the 0.05M acetate buffer (pH 4.7), enzyme, and water. Incubate the tubes at 30EC for 2 minutes. Start the reaction by the addition of sucrose. After exactly five minutes stop the reaction in the usual manner. Develop the color, determine the absorbance, and convert the absorbance readings into velocities.

#The easiest way to maintain these temperatures is to use a large beaker that is either heated on a hot plate or cooled by adding ice. It is not necessary to have these exact temperatures but the temperatures used should be close to these values and the actual temperatures recorded.

Determ ine the effect of an inhibitor on the invertase reaction by the following protocol.
*The sum of these three must be 1.5 mL and the amount of

To an assay tube add the 0.05M acetate buffer (pH 4.7), enzym e, and water. In a separate tube place approxim ately 1.5 m L of 0.3M sucrose. Place both tubes in a bath at one tem perature. After exactly

521 - ENZYM ES - Page 4 two m inutes of tem perature equilibration (do not exceed this tim e or denaturation m ay occur) transfer 1.0 m L of the sucrose to the assay tube. After 5 m inutes stop the reaction in the usual m anner. W hen all assays have been run develop the color and determ ine the velocity. Determ ine the effect of tem perature on the acid catalyzed hydrolysis of sucrose using the following protocol. #It is not necessary to have these exact temperatures, but the temperatures used should be close to these values and the actual temperatures recorded. In one tube place 2.0 m L of HCl and in another place approxim ately 1.5 m L of 0.3M sucrose. Incubate these tubes in a water bath m aintained at the appropriate tem perature. After 2 m inutes transfer 1.0 m L of sucrose solution to the HCl. After exactly 5 m inutes stop the reaction by the addition of 1.0 m L of 2N NaOH. Rem ove a 1.0 m L aliquot and develop the color using the usual procedure. It is again necessary to determ ine the velocity. This is accom plished in this case by m ultiplying the glucose concentration by 0.4. Four tenths is used in this case since the volum e is 4.0 m L. ENZYM ES Determination of Enzym e Concentration and Specific Activity Theory/Reaction W rite the reaction between sucrose and invertase. Results/Calculations Plot velocity versus m illiliters of dilute enzym e. Determ ine the slope which will be the concentration of the diluted enzym e. Calculate the concentration of the stock enzym e solution. Determ ine the protein concentration for the diluted and undiluted enzym e solution. Calculate the specific activity of the enzym e. pH Optimum of Enzym atic Hydrolysis Theory/Reaction Explain why enzym es exhibit a pH optim um Results/Calculations Plot velocity versus pH and determ ine the pH optim um of invertase. Results/Calculations Plot ln V vs. 1/T,K for both the enzym e and acid catalyzed hydrolysis of sucrose. Calculate the activation energy for invertase and acid catalyzed hydrolysis.

Determination of K m and V max, and Inhibition Theory/Reactions Explain the significance of K m and V max. Describe the three types of sim ple inhibition and explain how each affects a plot of 1/V vs. 1/[S]. Results/Calculations Using the data collected in the absence of inhibitor prepare plots of V vs. [S], 1/V vs. 1/[S], [S]/V vs. [S], and V/[S] vs. V. Calculate a value for K m and V max's. Using the data with inhibitor present plot 1/V vs. 1/[S] on the sam e graph as the data without inhibitor. Calculate the inhibitor constant(s) and determ ine the type of inhibition. Effects of Temperature on the Enzym e and Acid Catalyzed Hydrolysis if Sucrose Theory/Reactions Describe the effect of tem perature on a chem ical reaction and on an enzym e catalyzed reaction.

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