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Kinetics: Determination of an Enzymes Activity - Background

 

Studying the rates of enzymatic reactions and combining it with known information about an enzyme’s structure and catalytic mechanism(s) is an important tool for today’s biochemists. Through enzyme kinetics researchers can probe and identify a specific biological function of an enzyme and potentially suggest a way to modify its activity for a variety of industrial and medical purposes. Overall, kinetic assays of enzymes are performed to determine substrate specificity, enzyme performance (turnover), characteristics of the mechanism of action, and finally, fundamental kinetic constants describing the mechanism (vmax, KM, and kcat).

Enzymes catalyze an unfathomable number of reactions by using a combination of only six basic mechanisms: (1) acid-base catalysis; (2) covalent catalysis; (3) metal ion catalysis; (4) electrostatic catalysis; (5) proximity and orientation effects; and (6) preferential binding of the transition state complex. Independent of the mechanistic characteristics taken to generate product, the initial reaction rates of every enzyme can be analyzed in order to quantify their overall efficiency.

The initial reaction rates of enzymes are known to be affected by a variety of factors including enzyme concentration, substrate identity, substrate concentration, pH, temperature, and the presence, or absence, of known inhibitors. In this week’s experiment you will divide into groups to investigate the effects of these factors on invertase during the first part of the experiment, and then reconvene as individuals for the second part to study the effects of enzyme inhibition in greater detail and to re-test any experiments performed earlier in the week. Luckily, all of this is can be achieved from manipulating one equation—the Michaelis-Menten.


Michaelis-Menten I 


The Michaelis-Menten equation can be used to extract the maximal velocity (vmax), the Michaelis constant (KM), and the ratio of the catalytic constant (kcat), also known as the turnover number, to KM. In order to probe the characteristics of an enzyme of interest, scientists must quantify the values just mentioned by some sort of analysis. One of the easiest methods for determining such values is to invert the Michaelis-Menten:


Lineweaver-BurkCompetitive Inhibition

Commonly known as the Lineweaver-Burk or double-reciprocal plot, the interpretation of a graph like the one shown allows for the efficient determination of important kinetic values. Specifically, the data takes a linear form with the slope of the line equal to the ratio of KM to vmax and x- and y-intercepts of -(KM-1) and vmax-1 respectively.

Kinetic parameters are best estimated over a substrate concentration range of ~0.5 KM to ~5 KM. As a consequence, a majority of the measured data is limited to a small portion on the left side of the plot and is thus a major disadvantage of this analysis. In addition, when the substrate concentration is small, minor errors in quantifying the initial velocity will lead to greater errors along the y-axis and consequently dramatic errors in KM and vmax. It is important to realize that the Lineweaver-Burk is NOT the only type of plot utilized to quantify kinetic data. Each analysis has its advantages and disadvantages and with today’s technological advances, accurate determination of these values is now commonly done by computers utilizing sophisticated statistical treatments.

After observing the effects of enzyme concentration, substrate identity, substrate concentration, pH, and temperature on the activity of invertase, the second part of the experiment will be devoted towards enzyme inhibition. There are hundreds of substances that can alter the activity of enzymes by reversibly interacting with them in a manner that influences substrate binding and/or enzyme turnover. Specifically, these ‘substances’ are known as inhibitors, and in this part of the experiment you will be observing the effects of two known invertase inhibitors, copper (II) sulfate (CuSO4) and aniline (C6H5NH2).

Overall there are three different types of inhibition, competitive, uncompetitive, and noncompetitive:

1. COMPETITIVE INHIBITION

In this type of inhibition a substance directly competes with the normal substrate for binding to the enzyme’s active site. Competitive inhibitors, as they are termed, generally resemble the normal substrate so that it can specifically bind to the enzyme. However, enough difference exists between it and the substrate that its reactivity is completely different than the normal substrate. Therefore, these types of inhibitors reduce the concentration of free enzyme that is available for substrate binding.


UnCompetitive Inhibition

 

2. UNCOMPETITIVE INHIBITION

With uncompetitive inhibition, the inhibitor interacts directly with the enzyme-substrate complex but not to the free enzyme. In this case, the uncompetitive inhibitor, which does not have to resemble the substrate, renders the enzyme catalytically inactive by distorting its active site.


NonCompetitive Inhibition

 

3) NONCOMPETITIVE INHIBITION

During noncompetitive inhibition the inhibitor interacts with both the enzyme and the enzyme-substrate complex. Presumably, noncompetitive inhibitors are thought to bind to enzyme sites that participate in both substrate binding and catalysis and therefore affect both KM and vmax values.

It will be your job to summarize the optimal conditions for this enzyme and describe its inhibitory profile citing sufficient evidence to prove your claims!