In the Laboratory
Some Aspects of Yeast Anaerobic Metabolism Examined by the Inhibition of Pyruvate Decarboxylase Earl V. Martin Chemistry Department, Mount Saint Vincent University, 166 Bedford Highway, Halifax, NS B3M 2J6, Canada
Studies of metabolic activity in living cells generally require considerable prelaboratory preparation time and are often difficult to manage within a single three-hour student laboratory period (1, 2). Prerequisites for a suitable system to be used in such studies include having an easily maintained and ready source of viable cells that will accumulate, preferably outside the cell, a metabolite that is easily detected in low concentrations. This study describes such a system utilizing yeast cells under conditions in which pyruvate accumulates as a product of anaerobic glycolysis. Yeast cells (Saccharomyces cerevisiae) are readily obtained commercially as dry, active granules. These yeast preparations are stable in storage for reasonable periods, can be weighed with some accuracy, and, under proper anaerobic conditions, can be made to accumulate extracellular pyruvate from a carbon source such as sucrose or glucose (1). When oxygen is limiting, the normal fate of pyruvate in yeast is its conversion to acetaldehyde and carbon dioxide (3) by the catalytic action of pyruvate decarboxylase (EC 4.1.1.1): O CH3
C
O
O
C
O
–
CH3
pyruvate
C
H
+
CO2
acetaldehyde
The aldehyde is subsequently reduced to ethanol as the normal end product of glycolysis for this species under anaerobic conditions. The carbon dioxide generated in this reaction sequence is responsible for the leavening action of baker’s yeast (4). Pyruvate decarboxylase has optimum activity slightly below pH 7 (3) and shows much less activity under slightly alkaline (pH 8–9) conditions (1). The presence of inorganic phosphate has also been shown to have an inhibitory effect on pyruvate decarboxylase (5, 6 ). The combination of high pH and high phosphate concentration therefore causes the accumulation of pyruvate, which tends to be excreted into the medium (7 ). An assay using lactic dehydrogenase (EC 1.1.1.27), an enzyme not found in yeast, can then be used to measure the pyruvate accumulation. Lactic dehydrogenase, in conjunction with reduced nicotinamide adenine dinucleotide (NADH), detects pyruvate by conversion of this metabolite to lactate (8) according to the reaction O CH3
C
O C
pyruvate
–
O +
NADH
+
H
+
CH3
OH
O
CH
C
O– + NAD+
lactate
Extracellular pyruvate concentrations between 10 and 1000 nmol/mL can be reliably detected by means of this spectrophotometric assay, which measures the disappearance of NADH at 340 nm. In our introductory biochemistry course, students normally work in pairs to avoid delays with centrifuges and spectrophotometers. However, individual students or larger groups could be utilized. Within a single laboratory period each group
is easily able to develop a standard curve relating pyruvate concentration to absorbance change per minute and to answer simple questions such as, “Which sugars are most efficiently converted to pyruvate by yeast?” Experimental Procedure Baker’s yeast, most often Fleischmann’s Traditional, is obtained locally as the active dried granules. Lactic dehydrogenase from rabbit muscle, NADH, and sodium pyruvate standard solution (22.7 mM) are from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178-9916, USA. The working pyruvate standard (2.27 mM) is prepared by dilution of the above with 0.03 M phosphate buffer, pH 7.4. Lactic dehydrogenase enzyme solution to be used for assays is prepared by diluting 40 µL of the commercial suspension (10,000 units/mL) with 10 mL of the pH 7.4 phosphate buffer. This buffer is also used to prepare 0.002 M NADH for the assay. Disodium hydrogen phosphate, trichloroacetic acid (TCA), and sugars are aqueous solutions. Dry yeast granules (0.5 g) are placed in 20 × 200-mm tubes with 10 mL of aqueous solution containing up to 20% sugar, 0.1–0.5 M Na2HPO4, and any other compounds being examined. The pH at this point is slightly above 9. After various times of incubation (up to 15 min) at 37 °C, 4 mL of 10% TCA is added to curb metabolic activity and bring the pH back to neutrality. Cells are removed by centrifugation (10,000 rpm, 15 °C, 4 min) and a portion of the supernatant (usually 0.5 mL) is tested for pyruvate concentration. In lieu of centrifugation, cells can be removed by forcing small samples through 0.45-µm Millipore filters. The assay for pyruvate is carried out directly in a 1-cm (light path) cuvette containing at least 2.15 mL of pH 7.4 buffer, 250 µL of NADH solution, and 100 µL of dilute enzyme solution. The reaction is started by adding up to 500 µ L of working standard pyruvate or test solution, quickly mixing by inversion of the stoppered cuvette, and monitoring reaction progress using a Milton Roy Spectronic 1001 Plus UV–visible spectrophotometer. The kinetics program is set to take absorbance readings every 3 s for 15 s. The change in absorbance with time (∆A/min) for NADH oxidation by pyruvate is converted to pyruvate concentration by reference to a standard curve prepared by using known pyruvate solutions, up to 500 nmol, in the 3-mL reaction mixture (Fig. 1). Under conditions where higher pyruvate concentrations are developed, smaller sample volumes are employed while maintaining the final cuvette volume (3 mL) with buffer. Results and Discussion Pyruvate accumulation by S. cerevisiae, in the presence of sucrose for 10 min, is optimal at approximately 0.3 M HPO42᎑, which has a pH of 9.1 (Fig. 2). Pyruvate accumulation can be attributed mainly to the presence of a high concentration
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In the Laboratory
Figure 1. Standard curve. Kinetic data (∆A/min) related to known pyruvate concentration (nmol/mL sample) in the standard 3-mL reaction mixture. Oxidation of NADH is monitored for 15 s at 340 nm in the presence of lactic dehydrogenase.
Figure 2. Effect of phosphate concentration. Yeast pyruvate accumulation (nmol/mL medium) in 10% sucrose containing disodium hydrogen phosphate at various concentrations, pH 9.1.
of HPO42᎑, since results are the same whether the cations are sodium or potassium. In addition, similar pyruvate accumulation is not observed when 0.3 M carbonate at pH 9.1 is used to replace the phosphate anion. In 0.3 M phosphate at pH 9.1, pyruvate accumulation in the medium of the yeast cells is found to increase during a 15-min incubation. The accumulation of pyruvate can be directly related to the amount of sucrose present, reaching a maximum at about 2.5% sucrose after a 10-min incubation and remaining relatively constant up to 15% sucrose (Fig. 3). It is interesting for students to do a simple calculation based on the concentration of sucrose prescribed for the activation of yeast in most
bread recipes (4 ) and to discover that this corresponds to the amount of sucrose (ca. 2.5%) that gives optimum pyruvate production under the conditions used here. A simple experiment using this technique compares various sugars as the carbon source for effecting glycolytic pyruvate production; the incubation period is 10 min (Fig. 4). This clearly illustrates for students the immediate utilization of the glycolytic monosaccharides glucose and fructose for pyruvate synthesis. Enzymes for the conversion of galactose to glycolytic intermediates show no significant activity under the conditions employed. Similarly, the low rate of conversion of maltose and lactose to pyruvate indicates low levels of the
Figure 3. Effect of sucrose on pyruvate accumulation. Pyruvate concentration (nmol/mL medium) developed after incubation of yeast for 10 min in 0.3 M Na2HO4 containing various concentrations of sucrose.
Figure 4. Effect of carbon source. Yeast medium pyruvate (nmol/ mL) after 10 min in 10% sugar containing 0.3 phosphate, pH 9.1.
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In the Laboratory
hydrolytic enzymes (maltase and lactase) that convert these disaccharides to glucose. In contrast, yeast shows high levels of invertase (β-fructofuranosidase) for hydrolysis of sucrose to glucose and fructose. This enzyme permits the use of sucrose as the carbon source of choice for baker’s yeast (9). Each pair of students requires approximately 30 min of spectrophotometer time to do a standard curve for pyruvate and to experimentally answer one or two simple questions about yeast glycolysis. About half that time can be saved if a standard curve is supplied. Acknowledgments I wish to thank S. L. Boyd and R. S. McDonald for helpful assistance with this manuscript.
Literature Cited 1. Plummer, D. T. An Introduction to Practical Biochemistry; McGrawHill: London, 1978; p 311. 2. Robyt, J. F.; White, B. J. Biochemical Techniques, Theory and Practice; Brooks/Cole: Monterey, CA, 1987; p 253. 3. Holzer, H. Cold Spring Harbor Symp. Quant. Biol. 1961, 26, 277– 288. 4. McWilliams, M. Foods—Experimental Perspectives; MacMillan: New York, 1989; p 442. 5. Boiteaux, A.; Hess, B. FEBS Lett. 1970, 9, 293–296. 6. van Urk, H.; Schipper, D.; Breedveld, G.; Mak, P.; Scheffers, A.; Van Dijken, J. Biochem. Biophys. Acta 1989, 992, 78–86. 7. van Urk, H.; Mak, P.; Sheffers, A.; Van Dijken, J. Yeast 1988, 4, 283–291. 8. Kreig, A.; Rosenblum, L; Henry, J. Clin. Chem. 1967, 13, 196–203. 9. Dixon, M. Biochem. J. 1953, 55, 161–169.
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