A Study in Enzyme Kinetics Using an lon-Specific Electrode Sandra L. Turchi Millersville University, Millersville, PA 17551 Craig M. David University of Scranton, Scranton, PA 18510
John R. Edwards Villanova University, Villanova. PA 19085
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When studvine enzvme kinetics in an undereraduate bio" chemistry laboratory, one way the course of the reaction can he followed is hv monitoring a comoound aenerated concurrently with theknzyne-ca'&lyzed ieactio;. I n this series of experiments, the D-amino acid oxidase system is used. T h e oxidation of D-&in0 acids in the cell is accomplished by D-amino acid oxidase using molecular oxygen as the electron acceptor. Ammonia, HzOz, and an a-ketoacid are the products of the reaction. Catalase is added to the reaction media to destroy the Hz0z.l
+ pyruvate NH,
net
H,O
+ H,O-
+ XO, + D-alanine-
NHdt + OH-
pyruvate + NH,'
(1)
(2)
+ OH-
(4)
The oxidation can be followed by a variety of methods including the appearance of pyruvate (if alanine is the subby Oz electrodes, appearance of NH3 strate), decrease of 0% bv NHa electrode, or the disa~oearanceof the amino acid. his ex-periment uses the N H blectrode ~ since it is a simple andsensitive method. Since theelectrode issensitive to NH? and not NH4+,base has to be added to the aliquot to generate the NH3 molecule. This experiment is designed so the kinetics of the reaction can be followed. Experimental Preparation of NH3 Standard Curve Standardize the pH meter (Coming, Model 120) equipped with the NH3 electrode (Orion, Model 95-12) against NH&I (10-6-10-3 M). These solutions are prepared from a stock solution of NHlCl (0.100 M). Be sure that the final solutions have a pH > 12 (see p 5, Orion Man~al).~Plot the data on semilog paper with the concentration of NH3 on the log scale. The 0-140-mV scale gives the better measurement of the NH3 concentration. Sample Preparation Prepare solutions of D-amino acid axidase (1unit/mL) catalase (1000units/mL)and D-alanine (50 mM) inTris-HCI buffer (50 mM, pH 8).3 Combine the two enzyme solutions, and incubate at room temperature for 10 min. Rapidly mix the alanine solution (6 mL) with the combined enzyme solution (6 mL), and then immediately remove a l-mL aliquot. Pipette the sample into water (22 mL) and
sodium hydroxide (2 mL, 10 M). This will serve as the zero-time sample, so it is important to perform this step quickly. Measure the NH3 concentration with the ion-specific electrode. Take l-mL aliquots from the enzyme mixture incubation at 4-min intervaLs for 24 min. Add each aliquot to the alkaline solution, measure the NHa released, and then determine the NH3 concentration from the NH&l standard graph. Plot the formation of ammonia (in moles) vs. time. Determine how many micromoles of D-alanineare oxidized per minute by the enzyme preparation. Inhibitor Studies A Lineweaver-Burk double-reeiprical plot should also be prepared from which the V,, and K, for the reaction can he determined. Inhibition studies can also be performed in a subsequent laboratory period. Benzoic acid (sodium salt, 60 mM) and L-alanine (50 mM) can he tested as potential inhibitors of the D-amino acid oridase. Benzoic acid (1mL) or L-alanine (1mL) should be added to the combined enzyme mixture prior to the incubation at room temperature for 10min. The reaction is carried out as before (adjust the volume of the D-alanine solutions to 5 mL). The initial velocity should again be calculated and the data coplotted with the data from the K , determination on the Lineweaver-Burk plot. The apparent K,, V,,, and Kj can then he determined. Dlscuslon This experiment is a n easy way to introduce students t o enzymology. Both D-amino acid oxidase and catalase are stable enzymes. They are available from a number of reliable sources and do not need to be isolated from an organism. T h e determination of the Michaelis-Menten constant (K,) and a n investigation of potential inhibitors constitute the core of this laboratory. Among possible inhibitors, the benzoate ion (which inhibits) and L-alanine (which does not) are excellent choices, as they foster a discussion on the nature of the active site and the stereochemistry of chiral compounds.
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Acknowledament T h e authors wish to acknowledge the support of the Woodrow Wilson-Drevfus Summer Research Fund. We also want to thank ~ e n n i f e rFisher for her expert secretarial services.
' Decker. L. A,, Ed. Wmington Enzyme Manuat Freehold, KI,
1977. . . ~.
Orlon Manual; Drlon Research Inc.. Boston. MA. Dixon, M.; Kleppe, K. Biochim. Biophys. Acta 1965, 96, 383. Dixon. M.; Klepps. K. Biochim. Biophys. Acta 1965, 96, 369.
Volume 66
Number 8
August 1989
687
