Enzymes are proteins that act as biological catalysts that help increase the rate of nearly all

chemical reactions in living cells. They speed up reactions by providing an alternative reaction pathway
of lower activation energy and by stabilizing the transition state of reactions without changing the
chemical equilibrium between the products and reactants (University of Arizona, 1996). As catalysts,
they are not consumed and are not permanently altered by the reaction. Without the help of enzymic
catalysis, many biological processes necessary for life would be so slow that it would take years for them
to finish (Cooper, 2000). One such process in which enzymes play a very vital role is cellular respiration.

Cellular respiration is the process by which energy is released through the breakdown of the
chemical bonds of food molecules such as glucose (Nave, n.d.). In the breaking down of glucose, carbon
dioxide and energy in the form of adenosine triphosphate (ATP) are released. This reaction may proceed
in the presence of oxygen in aerobic respiration or anaerobically through the process of fermentation.

Aerobic reaction

C6 H12O6  6O2  6CO2  6H 2O  (16  18) ATP (1)

Anaerobic reaction (fermentation)

C6 H12O6  2C2 H 5OH  2CO2  2 ATP (2)

Baker’s yeast (Saccharomyces cerevisiae) was used to model the kinetics of the fermentation of
glucose. Yeast is an organism that can convert sugar into alcohol and carbon dioxide at both aerobic and
anaerobic conditions (Dashko, Zhou, Compagno, & Piškur, 2014).

The Michaelis-Menten kinetic equation provides one of the simplest and best-known approaches
to enzyme kinetics (University of Washington, n.d.). Published in 1913 by Leonor Michaelis and Maud
Menten, the equation relates the reaction rate to the substrate concentration. The chemical system is
shown below:


E  S  ES 
k1k2
 E  P (3)
k1

where 𝑘1 , 𝑘2 , and 𝑘3 are rate constants. In this system, a substrate (S) binds reversibly to an enzyme (E)
forming an enzyme-substrate complex (ES), which then reacts to generate a product (P) and to regenerate
the free enzyme (E). The rate law of the reaction is therefore given by:
𝑑[𝑃]
𝑟= 𝑑𝑡
= 𝑘2 [𝐸𝑆] (4)

Since [𝐸𝑆] is an unmeasurable quantity because it is an intermediate that is formed during the
reaction, a steady-state approximation can be made. In this assumption, the concentration of [𝐸𝑆] is low
and that it doesn’t change because the rate of its formation is equal to the rate of its breakdown. The
concentration of the free enzyme [E] is also unknown because some of the enzyme is bound (Indiana
University, n.d.). However, performing a site balance at [E] gives the following formula:

[𝐸] 𝑇 = [𝐸]0 = [𝐸] + [𝐸𝑆] (5)

Combining equation (5) with the steady-state approximation assumption to get the equation for
[𝐸𝑆] yields:
 [𝐸]0 [𝑆]
[𝐸𝑆] = (6)
𝐾𝑀 +𝑆

𝑘−1 +𝑘2
where 𝐾𝑀 = 𝑘1
and is the Michaelis constant. Substitution of equation (6) to equation (2) gives a new
form of the rate law:
𝑑[𝑃] 𝑑[𝑆] 𝑘2 [𝐸]0 [𝑆]
𝑟= = − = (7)
𝑑𝑡 𝑑𝑡 𝐾𝑀 +[𝑆]

The final form of the Michaelis-Menten equation can now be expressed as:
𝑉𝑚𝑎𝑥 [𝑆]
𝑟= 𝐾𝑀 +[𝑆]
(8)

In this form, 𝑉𝑚𝑎𝑥 = 𝑘2 [𝐸]0 , represents the maximum velocity (rate) achievable by the system at
maximum substrate concentration.

In order to fully characterize the kinetics of the enzymic reaction, integral and differential
analysis should be done on the Michaelis-Menten equation. In both methods, linear regression is
performed to the equation. The integral analysis involved the plotting of a certain concentration
function versus time curve of an experimental data (Wiley, Hepburn, & Levenspiel, 1964). Integration of
the Michaelis-Menten equation yields:
[𝑆]0 −[𝑆1 ] 𝐾2 [𝐸]0 𝑡
[𝑆] = [𝑆] + 𝐾𝑀 (9)
ln([𝑆]1 ) ln([𝑆]1)
0 0

In the differential analysis approach, the rate of expression is fit to the data directly without any
integration (Wiley et al., 1964). Since the Michaelis-Menton equation is nonlinear, manipulations should
be first done in order to linearize it. There are three available techniques to this: the Lineweaver-Burk,
Eadie-Hofstee, and Hanes-Woolf manipulations.

In the Lineweaver-Burk manipulation or the “double reciprocal plot”, the reciprocal of the rate is
plotted against the reciprocal of the substrate concentration. This manipulation allows for a quick
visualization of the different forms of enzyme inhibition. However, since this method uses the inverse of
the reaction rate, any small error could greatly affect the results obtained (Chemistry LibreTexts, 2017;
Dowd & Riggs, 1965).
1 𝐾𝑀 1 1
𝑟
= (𝑉 ) [𝑆] + 𝑉 (10)
𝑚𝑎𝑥 𝑚𝑎𝑥

For the Eadie-Hofstee plot, the manipulation was done by inverting the reaction rate and
substrate concentration and then multiplying them by 𝑉𝑚𝑎𝑥 . The advantage of this plot over the
Lineweaver-Burk manipulation is that it is more resistant against experimental errors. However, since the
values of the x- and y-axis of this plot are dependent on the reaction rate, any error done during the
experiment will propagate unevenly. The correlation for the goodness of fit is not applicable in this case
(Chemistry LibreTexts, 2017; Ranaldi, Vanni, & Giachetti, 1999). The rearranged form of this method is
shown below:
𝑟
𝑟 = −𝐾𝑚 (𝑠 ) + 𝑉𝑚𝑎𝑥 (11)
 Lastly, the Hanes-Woolf manipulation is obtained by multiplying the Lineweaver-Burk equation
[𝑆]
by [S] and by plotting 𝑟
versus [𝑆]. This plot is more preferred than the previous two because over a
wide range of [𝑆], the errors in the values of the y-axis provides a fair reflection of the errors in 𝑟
(Silverman, 2002). A drawback for this method is that both the abscissa and ordinate are dependent on
the reaction rate.
[𝑆] 1 𝐾𝑚
=𝑉 [𝑆] + (12)
𝑟 𝑚𝑎𝑥 𝑉𝑚𝑎𝑥

In this experiment, the kinetic modelling of the enzymatic fermentation reaction and the
determination of the kinetic parameters of the reaction was done through the integral and differential
analysis of the Michaelis-Menten equation. The effect of the glucose concentration and temperature on
the reaction rate was also observed. Furthermore, the process by which the reaction proceeded, whether
aerobically or anaerobically, was also determined.

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https://chem.libretexts.org/Textbook_Maps/Physical_and_Theoretical_Chemistry_Textbook_Map
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