Derek Wong
Biology 275L
Lab 5
Enzyme Kinetics of Beta-Galactosidase
Introduction
In order for reactions to occur, the activation energy of the uncatalyzed reaction must first be overcome. This energy barrier is called the free energy of activation and enzymes are catalysts that accelerate reactions by reducing the activation energy needed for the reaction to proceed.
Enzymes work by combining substrate with enzyme so that in the transition state, the enzyme-substrate complex would then disassociate to create the product and release the in-tact enzyme. Enzymes have several distinct characteristics. The first being that they are capable of lowering the energy of activation allowing for the reaction to proceed faster. Secondly, enzymes have a specificity allowing them only to bind to certain substrates at the active site(s). Enzymes can be regulated by feedback inhibition, covalent modification, or regulatory proteins.
Enzyme kinetics explain the relation between the rate and substrate at which the reaction occurs. Vmax is the rate at which the concentration of substrate becomes independent of the velocity of the reaction, in other words the maximum concentration of substrate is in the solution and adding more substrate will not have an affect on the reaction rate. This behavior is called the saturation effect and the reaction is a zero-order kinetics. Km is Michaelis constant which is the sum of the forward and backward reaction rate from the enzyme and substrate to the transition state and the reaction from the enzyme-substrate complex to the product. A sub-category of enzyme inhibition is competitive and non-competitive inhibition. Competitive inhibition means that there are other particles competing for the active site, affecting the Km, while non-competitive inhibition is when particles affect another site on the enzyme preventing the enzyme from binding. Non-competitive inhibition affects Vmax.
This lab will examine the enzyme kinetics of beta-galactosidase with and without an inhibitor. (Enzyme being beta-galactosidase; substrate being O-nitrophenyl-Beta-Delta-galactopyranoside [OPNG]; inhibitor being lactose). In this lab we want to find if there is competitive or non-competitive inhibition of the inhibitor. This is accomplished by analyzing the Lineweaver-Burk plot.
Discussion
The purpose of this lab was to create a Lineweaver-Burk plot to determine if the inhibitor, lactose, is a competitive or noncompetitive inhibitor. This was done by adding different substrate (o-nitrophenyl-Beta-Delta-galactopyranoside [OPNG]) concentrations to the enzyme, beta-galactosidase with and without the inhibitor. As the enzyme converts the substrate to product, the clear solution will turn yellow. Figure one shows the absorbance at 420nm readings for each substrate concentration versus time without an inhibitor. As the substrate concentration increased, the amount of absorbance was shown to increase in the spectrophotometer. In the first order region, the slope was taken. It was observed as the substrate concentration increased, the reaction rate (v) also increased. This is because the more substrate present, the more the product (the yellow color seen) may be made.
Figure two shows the absorbance at 420nm readings for each substrate concentration versus time with an inhibitor, lactose. Here the reaction rates were observed to be less and there seems to be a discrepancy in the substrate concentration of 0.08mM. This could be because too much substrate was added, or that when the substrate sample was taken, the vile containing it was not properly shaken so that there was substrate precipitate on the bottom of the vile.
Figure 3 shows the reaction rate (slope) versus substrate concentration with and without an inhibitor. The top curve represents the reaction rate without inhibitor, and the bottom curve represents the reaction rate for the substrate with an inhibitor. The reason why the substrate without inhibitor would be on top is because it would have a faster reaction. Without an inhibitor, the reaction can go faster when compared to a reaction with an inhibitor. If the lactose has a competitive inhibition affect, eventually both of the lines would merge into one line at zero-order kinetics since both would have the same Vmax value. If lactose is an non-competitive inhibition, then the two curves would have two distinct Vmax values.
Figure 4 is the Lineweaver-Burk plot, which is a way to confirm if lactose is a competitive or noncompetitive inhibitor. According to the plot, there is competitive binding meaning that the inhibitor binds to the enzyme’s active site so that the substrate can not bind and thus product is not produced (i.e. yellow color). Competitive binding can be overcome with the addition of more substrate, out competing the inhibitor. This is shown as both lines (substrate with and without inhibitor) crossed the y-axis. This also shows that although there are two different Km values (0.5 with inhibitor and 0.17 without inhibitor) for each line (Km being the x-intercept), both lines have the same Vmax value (since they both cross the y-axis at 0.0087).
The effects of competitive binding is that the Km are different for both the substrate with and without inhibitor, however the Vmax are still the same for both. The reason for this is because saturation will eventually occur even with the substrate with the competitive inhibitor. Eventually with enough substrate, the reaction will reach a zero-order. This can be related to as why in Figure 3, that both curves should intersect and merge since at Vmax they would be equivalent. The Km measures the enzyme’s affinity for the substrate and of course this will change as competitive inhibitor is added. The higher the Km the stronger the affinity and the lower, the weaker the enzyme’s affinity. If there are inhibitors the enzyme will not have the same probability of binding to the substrate.