Enzme Kinetics

Enzyme Kinetics

Email address:

SG: saida_guerra87@hotmail.com

Abstract

Background

Enzyme kinetics is the study of the rates of chemical reactions that are catalyzed by enzymes. The study of enzyme's kinetics provides insights into the catalytic mechanism of the enzyme, its role in metabolism, how its activity is controlled in the cell and how drugs and poisons can inhibit its activity (2). The rates of an enzyme catalyzed reaction generally changes with change in enzyme concentration, substrate concentration, temperature, pH, time, and presence of enzyme inhibitors. For this particular experiment, the determination of enzyme activity, Trypsin, was obtained by changing the concentration of its substrate concentration (1). For these purposes, the Michaelis-Menten equation was used in combination with a double-reciprocal or Lineweaver-Burke plot to plot obtained data and the enzymatic activity of Trypsin was measured through the colormetric Lowry's assay and spectrophotometer absorbance readings.

Results

End results of this experiment proved that the changes in substrate concentration are directly proportional to the rate of reaction of an enzyme. Looking at the graphs, it can be seen that an increase in substrate concentration would increase as the velocity of Trypsin increased. For instance, the initial velocity for the combination of test tubes 1 and 2 was .516 and the substrate concentration was .150, in the last recordings, for V and S, 2.236 and 2.252 were obtained, respectively.

Conclusions

The study of enzyme kinetics is sensitive to several factors, including temperature, pH, presence of inhibitors, and substrate concentration. In this particular experiment, results came out to prove that the greater the substrate concentration, the more substrate was converted into product by the enzyme.

Background

The term enzyme kinetics implies a study of the speed, rate or velocity of an enzyme catalyzed reaction, and of the various factors which may affect this. At the heart of any study of enzyme kinetics is knowledge of the way in which reaction velocity is altered by changes in the concentration of the enzyme's substrate and of the simple mathematics underlying this (2).

The reaction that is carried out by an enzyme can be represented by the following equation:

in which A is the Substrate, P is the product and E is the enzyme.

In an experiment carried out with a non-catalyzed chemical reaction, the velocity would be expected to have a direct relationship to that of the substrate concentration. In this case, the graph would be linear compared to a catalyzed reaction, which would happen to be a curve. If a person was to double the substrate concentration, then the velocity would also double. This is because if there is as twice as many substrates a molecule then the number which will have sufficient energy to undergo reaction will be doubled (2).

This is an example of a catalyzed reaction graph

Graph of velocity/[substrate]

The graph that is placed above is obviously not a straight line but a rectangular hyperbola and its equation is very complex, but is one of the things that will help derive many helpful and useful information from it. The equation shows v/[A] which is already known to be the work of Michaelis and Menton and by Briggs and Haldane, and it is usually called the Michaelis equation:

The two variables in this equation are the reaction velocity, v, and the concentration of substrate, a. V and Km are two constants which will require further explanation (2). From this equation, it can be seen how at low substrate concentration, the initial velocity, increases in an almost linear faction with an increase in substrate concentration. But, at higher substrate concentration levels, the velocity of the reaction increases only by small increments and then reaches a point of where there is no more measurable increase in velocity. This is when it is said that the reaction has obtained its Vmax, as can be seen in the graph below:

Then, scientist created the also known double-reciprocal or Lineweaver-Burke plots which plots the inverse of the velocity and the substrate concentration giving a linear graph of that of Michaelis-Menten, instead of a curve.

Lineweaver-Burke or double-reciprocal plot

Methods and Materials

Obtain 14 test tubes and label them 1-14. All the odd numbers use them as the control group and the even as the experimental. The casein is first added to test tube 1 and 2 with 0.1ml. Test tubes 3 and 4 had 0.2ml, 5 and 6 had 0.3ml, 7 and 8 had 0.4ml, 9 and 10 had 0.6, 11 and 12 had 1.0ml, finally 13 and 14 had 1.5ml. The Bicarbonate buffer was added to all test tubes as well but in all the control test tubes you added the enough ml in order to complete 3.0ml and in the experimental test tubes only 2.5ml were needed to reach. For instance, if you had 0.1ml of Casein in test tubes 1 and 2, then add 2.9ml in test tube 1 and 2.4 in test tube 2. Then start the reaction by adding 0.5ml of Trypsin only to the experimental tubes. Mix the content of tubes well on the vortex and incubate the tubes for 15 minutes at 37 degrees C, for the enzyme to act on the substrate. At the end of 15 minutes add 1.0ml of 15% TCA to each tube. Mix the tubes well on the vortex and keep the tubes at room temperature for 2 minutes. Centrifuge the tubes for 5 minutes. Very carefully, without disturbing the precipitated proteins a the bottom of the tubes, pipette out 1.0ml of the TCA soluble products in a new set of large sized test tubes which have been previously marked 1 through 14. Prepare a reagent blank tube containing 1.0ml of water and Lowry's reagent. Proceed by Lowry's Assay by adding 5.0ml to all tubes and wait for 10 minutes then add 0.5ml to all tubes and wait for 25 minutes. Adjust the