University of Lethbridge Department of Chemistry & Biochemistry

Chemistry 2740 Laboratory Experiment 1

A KINETIC STUDY OF THE EMULSIN ENZYME - CATALYSIS OF SALICIN

The hydrolysis reaction of salicin is
H HO HO H H CH 2OH H H O OH H O
CH 2OH + H 2O Catalyst

HO HO H

CH 2OH H H

CH 2OH O OH H + OH

OH

and is subject to catalysis by the enzyme emulsin (-D-glucoside glucohydrolase). The mode of action for the emulsin enzyme is the Michaelis-Menten mechanism. The substrate salicin [S] attaches itself at the active site of the emulsin enzyme [E] to form an enzyme-substrate complex [ES]. The [ES] complex dissociates to produce the product species [Pn] (n = number of products) and the original enzyme [E]. A more detailed discussion of the Michaelis-Menten mechanism and equation is not required for this experiment; instead we will postulate this simple model (1) for the single substrate reaction.
E + Salicin k k -1 Saligenin + E (1)

The rate law for the forward reaction is Rate = k[salicin][E] (2)

However, in this procedure, the salicin is greatly in excess of the enzyme emulsin. As a result, the enzyme is effectively saturated with the substrate. Therefore the addition of more substrate will not accelerate the reaction. Under these experimental conditions, virtually all the enzyme is present as an enzyme-substrate complex and the rate is independent of the salicin concentration and will now obey zero-order kinetics. The observed rate law becomes Rate = k (3)

In this experiment, the enzymatic hydrolysis of salicin by emulsin will be studied at two temperatures. This approach will allow for the determination of the activation energy (Ea) for the enzyme system. The change in temperature will increase the average energy of the salicin molecules which increases the likelihood of more reactant Page 1 - 1

Chemistry 2710 Laboratory Experiment 1 molecules possessing the required energy of activation. Therefore an increase in the reaction rate with increasing temperature allows for the calculation of the activation energy by the Arrhenius equation: k=AeEa/RT (4)

where k is the rate constant, A is a pre-exponential factor, R is the gas constant and T is the absolute temperature. It is important to realize that the activation energy (Ea) does not take into account entropy effects. As a result, a useful extension of this experiment is to calculate the entropy (S) using a derivation of the Wynne-Jones and Eyring equation: k =

k B T . n . S/R . E /RT e e e a h

(5)

where kB and h are, respectively, Boltzmanns and Planks constants. Gibbs free energy (G) and enthalpy (H) of activation can also be obtained by applying the following standard relationships G = H TS and Ea = H + RT (7) (6)

The method is a classic fixed time assay, which monitors the progress of the reaction spectrophotometrically. Aliquots of the reaction mixture are removed and quenched at specific intervals after the initiation of the reaction. The excess alkali used to halt the reaction also serves to quantitatively convert the saligenin product to its anion (8), which has a max at 290 nm.
CH 2OH OH CH 2OH O

Base

(8)

The hydrolysis reaction of salicin is also subject to acid catalysis. The latest literature Page 1 - 2

Chemistry 2710 Laboratory Experiment 1 value for the activation energy Ea is around 23.6 kcal/mole for the acid catalyzed reaction and is reported here for comparison purposes. Apparatus UV Spectrophotometer (Cary1E Spectrophotometer or Ultrospec 1000); pH meter and calibration buffers; 25oC and 37oC water baths; vortex mixer; Eppendorf pipet, Brinkman dispenser and timer. The operation of the Cary 1E or the Ultrospec 1000 will be demonstrated by your instructor. Reagents Emulsin III, -D-glucoside glucohydrolase EC 3.2.1.21 from Sigma Chemical Company; Sigma brand salicin, (2-[Hydroxymethyl] phenyl--D-glucopyranoside); NaOH pellets. Waste Disposal A 4-liter bottle for the collection of wastes is supplied with the experimental set up. All excess stock reagents, unused reaction solutions and solutions from the reaction vessel should be disposed of in this bottle. The glassware can then be given a single small rinse into the waste container before being cleaned further in the sink. In preparing reaction solutions, only remove as much reagent from the stock container as is necessary to make the reaction mixtures. Procedure Two kinetic experiments will be conducted; one at 25oC and the other at 37oC. The resulting rate constants at the two temperatures will be used to calculate the activation energy, Ea and the pre exponential factor A of the Arrhenius equation for this reaction. Prepare 100 mL of 0.1 M sodium phosphate buffer, pH 6.1 + 0.2 [refer to Appendix C to read how to prepare this buffer]. Weigh accurately 3.5 mg of emulsin into a clean and dry 25 mL Erlenmeyer flask. Dissolve the enzyme in a 10 mL aliquot of 0.1 M phosphate buffer, pH 6.1 + 0.2. Weigh accurately 135 mg of the salicin substrate into another clean dry 50 mL Erlenmeyer flask and dissolve in 20 mL aliquot of the same buffer.

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Chemistry 2710 Laboratory Experiment 1 It is imperative that the stock solutions and glassware (reaction vial, 5 and 10 mL Moir pipettes) be properly thermostated. Place the stock solutions of emulsin and salicin in the 25oC bath and allow sufficient time for thermo equilibration. Prepare a set of 14 labelled 18 x 150 mm test tubes each containing 10 mL of approximately 2 M NaOH. Please note that this NaOH solution is very caustic and must be handled with extreme care. When thermo equilibrium is achieved the reaction is started as follows: a 2.5 mL aliquot of the emulsin enzyme is transferred to a reaction vial previously charged with 7.5 mL of the salicin substrate. Quickly and thoroughly mix the contents and return the vial to the water bath. The time is noted, and a 300 L sample of the reaction mixture is immediately removed and dispensed into the first labelled test tube. Mix the contents of the test tube with the aid of the vortex. Thereafter 300 L aliquots are removed at intervals of exactly 3 minutes until a total of 7 test tubes are used. The entire process is repeated at 37oC using the remaining 7 test tubes. The absorbance at 290 nm is read for all the tubes against a blank of 7.5 mL 2 M NaOH and 2.5 mL distilled water.

Before leaving the laboratory, please enter names, date, and experimental data into
the computer. Calculations Calculate the apparent zero order rate constants for the two experiments by preparing a plot of the absorbance against time. The slope of the straight line obtained is the zero order rate constant. Calculate the activation energy, Ea, and the pre-exponential factor, A, of the Arrhenius equation (4) for this reaction. This is accomplished by first using the following equation to solve for Ea and then using this value along with equation (4) to solve for A. k E ln 2 = a 1 1 k1 R T2 T1 (9) Calculate the entropy, S, using equation (5) (assume n=1), and the free energy of activation (G) and enthalpy (H) using equations (6) and (7).

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