Adam Capriola

Biology Lab Section 109

Dr. Ratterman

10/22/06

Enzyme Kinetics Lab

The Relationship Between Enzyme and Substrate Concentrations and Rates of Reaction

Abstract

Enzymes are catalysts which lower the activation of chemical reactions, thus making them happen more rapidly. In this experiment, different amount of enzyme and substrate were put in a test tube, then were observed using a spectrophotometer to see how fast the reacted to produce product. It was found that as the concentration of enzyme was increased, the speed of reaction increased. Likewise, it was found that as the concentration of substrate was increased, the speed of reaction increased. As there is more enzyme, it is able to react with more substrate at once, therefore increasing the rate of reaction. When there is more substrate, there will be just as much enzyme, also making the rate of the reaction increase. Thus, higher levels of enzyme or substrate mean there will be a higher turnover rate of product.

Introduction

A catalyst is a substance that lowers the activation energy of a chemical reaction (Zubay et al., 1995). In order words, this means that a catalyst makes chemical reactions happen faster than they normally do. Enzymes are catalysts that speed up reactions in living cells. For example, the enzyme carbonic anhydrase makes the chemical reaction CO2 + H2O ---> H2CO3 happen 107 times faster than it normally does. In this example, the enzyme reacted with CO2. The substance which an enzyme catalyzes is called a substrate. Enzymes bind to substrates in order to speed the reaction in turning the substrate to a product. When there is a little amount of substrate, there will be a small amount of enzyme, but as the level of substrate increases, the level of enzyme increases. In this experiment, the speed at which enzymes and substrates react was observed.

Materials and Methods

For the first experiment, different enzyme concentrations were tested to see how fast they reacted. A Spec20 spectrophotometer was first turned on and set to a wavelength of 340 nm. A reaction mixture consisting of 0.850 ml distilled water, 1.000 ml buffer stock, 0.100 ml 15 mM NAD+, and 1.000 ml ethanol was then put into the test tube using a micropipettor. This mixture was agitated to mix it together, and the spectrophotometer was then calibrated using this test tube. Once calibrated, 0.050 ml ADH enzyme was added to the test tube mixture and agitated. The test tube was then quickly put into the spectrophotometer and its absorbance readings were recorded. The readings were recorded every 15 seconds for 3 minutes. This process was repeated for 2 more reaction mixtures with adjustments to the amount of distilled water and AHD enzyme included. The second reaction mixture had 0.100 ml ADH enzyme and 0.800 ml distilled water, and the third one had 0.025 ml ADH enzyme and 0.875 ml distilled water.

For the second experiment, different substrate concentrations were tested to see their reaction rates. Once again, a Spec20 spectrophotometer was turned on and set to a wavelength of 340 nm. A reaction mixture consisting of 1.85 ml distilled water, 1.0 ml buffer stock, 100 ul NAD+, and 0 ul ethanol stock was put into a test tube using a micropipettor. The mixture was agitated, then put into the spectrophotometer and the spectrophotometer was calibrated. Once 50 ul of ADH was added to the reaction mixture, the test tube was agitated and quickly put into the spectrophotometer. Absorbance readings were then recorded every 15 seconds for 2 minutes. Another reaction mixture with the same amounts of each substance was made, observed, and recorded. This process was repeated for five more reaction mixtures with differences in the amount of distilled water and ethanol stock used. The amounts of distilled water and ethanol stock for these reaction mixtures were 1.80 ml distilled water and 50 ul ethanol stock, 1.75 ml distilled water and 100 ul ethanol stock, 1.65 ml distilled water and 200 ul ethanol stock, 1.35 ml distilled water and 500 ul ethanol stock, and 0.85 ml distilled water and ethanol stock.

Results

Table I

Time (min:sec) / Absorbance (50 ul enzyme) / Absorbance (100 ul enzyme) / Absorbance (25 ul enzyme)
0:00 / n/a / n/a / n/a
0:15 / n/a / n/a / n/a
0:30 / n/a / .15 / .035
0:45 / .14 / .22 / .07
1:00 / .175 / .28 / .095
1:15 / .21 / .3325 / .12
1:30 / .24 / .38 / .145
1:45 / .27 / .42 / .1675
2:00 / .2975 / .455 / .19
2:15 / .32 / .485 / .2125
2:30 / .3425 / .505 / .235
2:45 / .36 / .525 / .255
3:00 / .3825 / .55 / .2725
3:15 / .4 / .57 / .29

Table II

Amount of Enzyme (ul) / [E] (ug/ml) / Initial Slope (A/min) / Velocity (umol/min)
50 / 0.00333 / 1.60 / 0.772
100 / 0.00667 / 2.29 / 1.10
25 / 0.00167 / 0.889 / 0.429

The concentration of enzyme had an effect of the reaction rate. As the concentration of the enzyme went up, the velocity went up (Table II, Figure 2). The initial slope also became steeper as the concentration of the enzyme increased (Table II, Figure 1).

The concentration of substrate affected the rate of reaction. As the concentration of substrate went up, the velocity increased steeply then evened out (Figure 4). From Figure 4 (v vs. [S]), the estimated Vmax is 0.0620 umol/min and the estimated Km is 0.0140 mM. From Figure 3 (1/v vs. 1/[S]), the estimated Vmax is 0.0568 umol/min and the estimated Km is 10 mM. The actual Vmax and Km recorded from SigmaPlot were 0.06566 umol/min and 0.01522 mM. The Vmax obtained from each graph are fairly close, but the Km are not very close at all. The Vmax and Km from the v vs. [S] graph are very similar to the actual Vmax and Km obtained from SigmaPlot.

Discussion

Shono and co-workers (1995) observed the rate of reaction versus the concentration of a substrate different from the one used in this experiment. Their graphs resulting from their experiment are very similar to the graph resulting from this experiment. The “velocity vs. substrate concentration” graphs follow the almost exact same curve, but the levels of concentration were higher in Shono’s experiment, resulting in higher rates of reaction. In the “1/v vs. 1/[S]” graphs, Shono’s line seems to have about the same slope, but it crosses the x-axis at a much lower value than it did in the graphs for this experiment.

This can be attributed to error in procedure of the experiment, which caused outlier values the line to be skewed. It was not known these outlier values should have been disregarded until computing calculations for Km and Vmax using the “1/v vs. 1/[S]” graph and comparing to other similar graphs. The Vmax recorded from Figure 3 was fairly close to the Vmax found from Figure 4. The Km from Figure 3, 10 mM, is not very close, however. That value is way larger than the Km found from Figure 4, 0.0140 mM, and the actual Km calculated by SigmaPlot, 0.01522 mM.

From the experiment, the results showed that with a higher concentration of enzyme, the higher rate of reaction. When catalyzing a reaction, the enzyme binds to the substrate (Bolsover et al., 1997). If there is a higher concentration of enzyme, this means that there will be more enzyme to bind to substrate at once, therefore making the turnover rate of substrate to product higher. This is why a higher concentration of enzyme produces a higher turnover rate. The turnover rate begins to slow down and stop as the amount of substrate runs out, and that is why the absorbance rates began to even out in Figure 1. There was a limited amount of substrate that could be converted into product.

As there was a higher concentration of substrate, the rate of reaction increased and then leveled off, as shown in Figure 4. The Michaelis-Menten graph assumes that the enzyme and substrate are in equilibrium (Zubay et al., 1995). Therefore, as there is a higher concentration of substrate, there will be an equally high concentration of enzyme to react with the substrate. As there is a higher concentration of each, the rate of reaction increases. The curve evens off, however. This is because each substrate has a maximum velocity at which it can convert from substrate to product. The enzyme can not catalyze the substrate to turnover faster than this.

The affinity for the enzyme and substrate in this experiment was fairly high. The Km, 0.01522 mM, is a low number. This means the enzyme and substrate reacted very quickly to produce product.

Literature Cited

Bolsover, S.R., J.S. Hyams, S. Jones, E.A. Shepard, and H.A. White. 1997. From Genes

to Cells. (Wiley-Liss, NY). 424 p.

Shono, M., M. Wada, T. Fujii, 1995. Partial Purification of a Na+ -ATPase from the

Plasma Membrane of the Marine Alga Heterosigma akashiwo. Plant Physiol 108:

1615-1615.

Zubay, Geoffrey, William M. Parson, and Dennis E. Vance. 1995. Principles of

Biochemistry. (Wm. C. Brown, Dubuque, Iowa) 863 p.