Enzyme Function

Introduction

In this experiment, the lactate dehydrogenase-catalase reduction of pyruvate to lactate in the presence of reduced nicotinamide adenine dinucleotide (NADH) as the coenzyme, was studied. When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated from NADH by the reduction of pyruvate to lactate. Some tissues and cell types (such as erythrocytes), which have no mitochondria and thus cannot oxidize pyruvate to CO2, produce lactate from glucose even under aerobic conditions. The reduction of pyruvate is catalyzed by lactate dehydrogenase (LDH), which forms the L-isomer of lactate at pH=7:

CH3-CO-COO- + NADH + H+ ® CH3-CH(OH)-COO- + NAD+

NADH has an absorption maximum at 340 nm. So the reaction was followed spectrophotometrically, by observing the decrease in absorbance at 340 nm. In this experiment, the Michaelis constant was determined and the inhibitory effect of oxamate was demonstrated.

Procedure

Part A: Without inhibitor

A series of eight tubes were prepared as shown at table 1:

Tubes # 1 2 3 4 5 6 7 8

Imidazole HCl buffer, pH=7.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

NaPyruvate, 1 mM 3.0 3.0 2.0 1.2 0.8 0.6 0.4 3.0

H2O 0.8 0.7 1.7 2.5 2.9 3.1 3.3 1.2

NADH,1mM 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0

Enzyme (~10 mg/ml) 0 0.1 0.1 0.1 0.1 0.1 0.1 0.1

The tubes were prepared without the addition of the enzyme LDH (lactate dehydrogenase).

The spectronic colorimeter was standardized at 340 nm and the absorbance was set to read zero against tube 8. A zero time value was obtained by reading tube #1, Abs=0.645. After standardization 0.10 ml of LDH was added to tube #2 as timer was activated, the tube was sealed with parafilm and the reactants were mixed by inverting the tube. Then the tube was placed in the spectrophotometer. The reaction was followed by recording the time, each time that the needle passed over a set line on the absorbance scale. This was repeated over a total of three minutes. The procedure was repeated for each tube one at a time.

Part B: Inhibition by oxamate (H2N-CO-COO-)

The procedure, as above, was repeated with the addition of 0.1 ml of 0.2 mM Na oxamate to each tube, as shown in table 2. The oxamate was added prior to the addition of the enzyme. In order to keep the total volume at 5.2 ml, 0.1 ml less water was added to each tube.

Table 2

Tube # 1 2 3 4 5 6 7 8

Imidazole HCl buffer, pH=7.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

NaPyruvate 1mM 3.0 3.0 2.0 1.2 0.8 0.6 0.4 3.0

H2O 0.7 0.6 1.6 2.4 2.8 3.0 3.2 1.1

NADH 1mM 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0

Inhibitor (0.2mM Na Oxamate) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Enzyme (~10 mg/ml) 0 0.1 0.1 0.1 0.1 0.1 0.1 0

Observations and Results

Part A: Without inhibitor

The absorbance values, which were obtained from each tube for every 15" are illustrated in table 3. For each tube, from #2-7, the absorbance (y-axis) is plotted versus time (x-axis), using the absorbance read in tube #1 for the zero time for all lines. The six lines are shown in figure 1.

Table 3

Tube 15" 30" 45" 60" 75" 90" 105" 120" 135" 150" 165" 180"

2 0.395 0.280 0.215 0.150 0.092 0.049 0.024 0.018 0.017 0.017 0.017 0.017

3 0.375 0.325 0.271 0.218 0.156 0.107 0.064 0.044 0.032 0.027 0.025 0.024

4 0.358 0.314 0.267 0.225 0.181 0.144 0.109 0.077 0.047 0.029 0.019 0.015

5 0.496 0.415 0.378 0.344 0.312 0.278 0.254 0.227 0.201 0.176 0.153 0.136

6 0.386 0.354 0.324 0.299 0.273 0.249 0.225 0.203 0.181 0.162 0.142 0.126

7 0.384 0.365 0.345 0.324 0.304 0.287 0.270 0.255 0.239 0.225 0.212 0.198

The slope (m) for each of the lines in plot in figure 1, are shown in Table 4. Table 4 also illustrates the concentration of pyruvate, 1/[pyruvate], the velocity and 1/velocity. The velocity in each tube, was calculated by multiplying the slope of each tube with 6.22x10-3, which is the molecular extinction coefficient of NADH. According to table 4, a graph of the concentration of pyruvate vs. the velocity is plotted and is shown in figure 2.

Table 4

Tube [pyruvate] (μΜ) 1/[pyruvate] (μM-1) m (slope) (Δabs/min) Velocity (Δ[ΝΑDΗ]/min) 1/Velocity

2 577 1.7x10-3 -0.0028 1.742x10-5 5.74x104

3 385 2.6x10-3 -0.0029 1.804x10-5 5.54x104

4 231 4.3x10-3 -0.0028 1.742x10-5 5.74x104

5 154 6.5x10-3 -0.0024 1.493x10-5 6.69x104

6 115 8.7x10-3 -0.0021 1.306x10-5 7.66x104

7 77 13.0x10-3 -0.0017 1.057x10-5 9.46x104

Figure 2: The Michaelis-Menten plot with and without inhibitor.

In figure 3 are illustrated the Lineweaver-Burk graphs with and without inhibitor. The Michaelis-Menten equation is the following:

V0= (VmaxЧ[S])/(Km+[S])

Where Vmax is the maximum velocity, V0 is the initial velocity, [S] is the concentration of the substrate (pyruvate) and Km is called the Michaelis