Determining Optimum Environmental and Chemical Effects on Enzyme Kinetics

Abstract

Enzymes, fundamental proteins that catalyze reactions by lowering activation energy, operate under kinetic principle and can react slower or faster with different ph and temperature levels. By finding this optimum temperature and pH through a process of spectrophotometery we can understand how many of the enzymes in our body function in different environments. I will examine a set of pH ranges and temperature ranges to find which one has the fastest reaction rate due to amount absorbed and transmitted as light is passed through the solution. The graph of the absorbances vs. time will provide for the calculation of the reaction rate from the best fit curve and thus showing where the optimum level is. This was found to be at 60o C and a pH of about 5 where there was more product formation in the shortest interval. As temperature increases the rate, we see how this would produce the best range for temperature from molecular kinetics and a balance of charge and equilibrium from H+ ions in pH 5 would give the best performance. Extremes of both though would hinder the formation of the enzyme and harm the active site along with the interaction between substrate and enzyme.

Introduction

Proteins have many functions in living organisms. One of the most important of these functions is catalyzing reactions through a protein structure called the enzyme. The enzymes, like all proteins, are composed of linked amino acids connected by hydrogen bonds and folded into functional shapes with the help of water. The enzyme lowers the activation energy of a reaction, which is the amount of energy required for a reaction to occur; without enzymes many reactions would not have the necessary energy to take place .The reaction rate between the reactant, or the substrate, and the enzyme is based upon kinetic principles and is thus dependant on environmental and chemical factors such as temperature, ph and concentration. Substrate concentration plays a major role in rate of reaction with lower substrate concentrations exhibiting first-order kinetics while large substrate concentrations not being able to bind to active sties as quickly, reduce the reaction rate overall. (Kivela Tillotson 2007)

The optimum temperature and pH in which reaction rate is the greatest, or there is more product formation, can help biochemists efficiently produce products and understand how many of the enzymes in our body function over different temperature and ph ranges. Enzyme immobilization, by operating outside of the optimal range, can allow for enzymes to operate much longer and offer many economical advantages (Woster). Dr. Maud Menten and Dr. Leonor Michaelis did extensive research in this and developed a rate constant showing the affinity for the substrate by the enzyme. They found out that a change in ph will cause a shift in equilibrium and therefore change the change the shape because of the change in charge. (Garrido del Solo, & Garcia Canovas, 1995)

I will use the enzyme О±-amylase, a byproduct of barley plant, to determine the optimum temperature and pH range using the substrate starch, by the process of spectrophotometery. Because absorbance is directly proportional to the concentration of color in the solution, the amount of products formed can be measured from amount of light absorbed or transmitted. Starch is a colorless solution so the addition of iodine will from a colored complex that can be measured. The graph of enzymatic reaction as a function of pH should produce a bell-shaped curve due to equilibrium change of H+ ions (Koning 1994). Temperature should show a similar graph due to the low kinetic speeds at low temperatures while thermal denaturation at high temperatures.

Material and Methods

Materials:

О±-amylase solution, starch solution , I2KI solution, Erlenmeyer flask, Spectrometer, Distilled water, 11 cuvettes, Ph buffer solutions range 4-6, micro-pipette, graduate cylinder, temperature bath.

Method:

In order to measure the rate of product formation in relation to time I used a spectrophotometer to measure absorbance of light passing through the solution. The О» max, or the wavelength of light at which the substance has maximal absorbance, was determined to be 560 nm by testing the starch and iodine solution over a range of wavelengths. The enzyme used, О±-amylase,