Biochemistry Lab Report: Lactate Dehydrogenase Enzyme Kinetics Study

This lab report details an experiment on the kinetics of lactate dehydrogenase (LDH), an enzyme crucial in glycolysis, which catalyzes the reduction of pyruvate to lactate in the presence of NADH. The study aimed to determine the Michaelis constant (Km) and the effects of inhibitors on the reaction. The experiment involved analyzing the enzyme's behavior under various conditions and plotting data to derive the Lineweaver-Burk equation. The report discusses the three types of inhibition: competitive, non-competitive, and uncompetitive, and how they affect the reaction rate. The findings are based on data analysis, including the creation of four graphs, to satisfy the laboratory requirements and objectives. The report references relevant sources and provides a comprehensive overview of the experiment's methodology and results.

Running: Biochemistry
Biochemistry
Institution Name
Student Name
Date of Submission
1

Biochemistry
Abstract
Glycolysis a reaction which takes place in the cytoplasm and is the first step in the
breakdown of glucose to extract energy for cell metabolism. It functions with either aerobic
conditions or anaerobic conditions. Substrate level phosphorylation verses oxidative
phosphorylation produce ATP during glycolysis process for example ADP +
phosphoenolpyruvate→ATP + pyruvate. One redox reaction occurs during glycolysis whereby
NAD+ accepts the electrons during oxidation which leads to its reduction and 2NADH are
produced (Saupe). Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate
with concomitant interconversion of NADH and NAD+ .It coverts pyruvate, the final product of
glycolysis to lactate when oxygen is absent or in short supply.
The purpose of this experiment was to study the kinetics of dehydrogenase-catalyzed
reduction of pyruvate to lactate in the presence of NADH as the coenzyme. The experiment
involved two processes whereby the first process involved determination of Michaelis constant
of the reduction reaction of NADH with LDH and the next process involved determination of the
effect of inhibitors in the reaction process.

Biochemistry
Introduction
Michaelis Menten equation was obtained by applying the steady sate approximation to
the mechanism below,
Michaelis Menten equation became;
V = k 1 [E]tot [E ]
Km +[S ] = V max +[S]
Km+[ S]
Where [ E]tot the total amount of enzyme is present in the assay and Km is Michaelis constant
given by;
Km = kcat + k−1
k1
The equation was derived for a single substrate –single product reaction, this formula was
still valid for LDH with a condition that substrates was held at constant initial concentration. A
better way of determining Vmax and Km was by plotting the reciprocal of Michaelis Menten
equation which was given by Lineweaver-Burk equation shown below;
This is a linear equation which gave a straight line with 1
V max
as y-intercept and Km
V max
as
its slope.
Apart from determination of Km and V max , Lineweaver-Burk equation allowed
determination of the three types of inhibition behaviors and each type is distinct and results in an
altered rate law as shown;

Biochemistry
Competitive
Non-competitive
Uncompetitive
After plotting the equations, competitive inhibition is confirmed if the linear regression of
the reaction with presence of inhibitor is intersecting with the reaction with no inhibitor on the
1
V max
meaning there is direct competition between the inhibitor and substrate for the enzyme
binding site (Chenault & Whitesides, 1988). If both of the linear regression are parallel to each
other, the inhibitor binds to the ES complex not the enzyme hence it is an uncompetitive
inhibition. Mixed inhibition occurs if the inhibitor binds to both enzymes alone and ES complex
and both regression lines intersect on the left quadrant.
After the experiment, data was recorded and used to draw four graphs which were then
analyzed and discussed in detail to satisfy the requirements and objectives of the laboratory
experiment. This lab report was written after reading widely on the topic of study on the relevant
textbooks.

Biochemistry
References
Chenault, H. K., & Whitesides, G. M. (1988). Lactate Dehydrogenase-Catalyzed Regeneration
of NAD from NADH for Use in Enzyme-Catalysed Synthesis. Massachusetts: Havard
University .
Saupe, S. G. (n.d.). Introduction to Cell and molecular Biology. Collegeville: College of St.
Benedict John's University .