"Cell Growth Due to Mutations in DNA

for Enzymes of Cellular Respiration"

Jason Trimble

Red 1

Dr. Thiemann

Introduction

All living organisms must have energy to survive and carry out their daily activities.

Most of this energy comes in the form of adenosine triphosphate (ATP). The organisms harness

this energy from ATP through the process of cellular respiration. In this process, food molecules,

such as glucose, are oxidized in the presence of oxygen and water to carbon dioxide and more

water (C6H12O6 + 6O2 + 6H2O -> 12H2O + 6CO2). ATP is another product that is produced. The

steps of this process are glycolysis, the oxidation of pyruvate to acetyl CoA, the Krebs cycle, and

oxidative phosphorylation ("Cellular Respiration"). All but glycolysis occur in the mitochondria

organelles of the cell. Specifically in glycolysis and the Krebs cycle, substrate-level

phosphorylation produces ATP when enzymes bring substrates and ADPs together. In glycolysis,

a glucose molecule is broken down into two pyruvate (C3H3O3) molecules. This process requires

no oxygen and takes place in the cytosol of the cell. The glucose molecule is modified by a chain

of 10 enzymes with the end products being 2 pyruvate molecules, 2 net ATP (2 ATP are used to

make 4 ATP), and two reduced NADH electron carriers ("Glycolysis"). In the small oxidation of

pyruvate step, pyruvate enters a mitochondrium, 1 CO2 molecule is released, a NADH electron

carrier is reduced, and pyruvate is converted to acetyl CoA. In the Krebs cycle, all of the carbons

are released as CO2 in an 8-enzyme cycle. For each pyruvate that enters the cycle, 1 ATP, 3

NADH, and 1 FADH2 are generated. The three experiments done on 7 different strains of a new

eukaryotic single-celled organism with mutated genes helps determine what the phenotype of

each gene is. Each mutated gene encodes for a certain enzyme in either glycolysis or the Krebs

cycle. If the mutation affects the function of the enzyme, the typical substrate of the enzyme will

accumulate in the cell by using Nuclear Magnetic Resonance and other techniques. However, the

different phenotypes of the mutated genes are only apparent at a higher "restrictive" temperature

(37 C), and all the cells appear normal or wild-type at a lower "permissive" temperature (25 C).

Since there is a mutation in the genes of each strain, they should all grow at 25 C but also all not

grow at 37 C when provided with the starting molecule of glucose which is the first experiment.

When given pyruvate in the second experiment, a certain strain should grow if the mutation is in

a gene that encodes for an enzyme of glycolysis because this part of cellular respiration has been

skipped. Otherwise, the strain should not grow since there is either a mutation in a gene for an

enzyme involved in the oxidation of pyruvate or the Krebs cycle. For the third experiment with

fatty acids as a food source, the three strains used should grow since this uses a separate

metabolic pathway than glycolysis and the Krebs cycle. The fatty acids are used in acetyl CoA,

so this bypasses glycolysis and the oxidation of pyruvate in cellular respiration.

Results

Growth of Mutated Strains on Glucose

Strain 136 152 164 166 189 195 211

Growth at

25 C

+ + + + + + +

Growth at

37 C

- - - - - - -

Substance

Remainin

g

Fructose 1,6 bisphosphat

e

3 - phospho

-glycerat

e

pyruvate

pyruvate

Phosphoenol-pyruvate

2 - phospho

-glycerat

e

pyruvate

Figure 1: Feeding Glucose to Mutated Strains. The mutated strains were grown both at the

permissive temperature of 25 C and the restrictive temperature of 37 C on glucose. The

chemicals that accumulate at the restrictive temperature were found out by using Nuclear

Magnetic Resonance and other chemical techniques.

Growth of Mutated Strains on Pyruvate at 37 C

Strain 136 152 164 166 189 195 211

Growth + + - - + + -

Figure 2: Feeding Pyruvate to Mutated Strains. The mutated strains were grown on pyruvate only

at the restrictive temperature of 37 C.

Growth of Mutated Strains on Fatty Acids at 37 C

Strain 164 166 211

Growth + + +

Figure 3: Feeding Fatty Acids to Mutated Strains. The three strains(164, 166, 211) that have not

grown yet at the restrictive temperature of 37 C were grown on fatty acids with the other

conditions remaining the same.

Enzymes Affected by the Mutations in Each Strain

Strain 136 152 164 166 189 195 211

Enzyme

Affected

Aldolase Phospho-

glyceromutas

e

Pyruvate

dehydrog-

enase

Pyruvate

dehydrog-

enase

Pyruvate

kinase

Enolase Pyruvate

dehydrog-

enase

Figure 4: The Mutated Enzymes for Each Strain. The enzymes affected by the mutations in each

strain were deduced by using the fact that the substance that accumulates from Figure 1 is the

substrate for the enzyme.

When growing the strains on glucose in experiment 1, only the restrictive temperature

makes the phenotypes of the mutations appear. All mutated strains grew at 25 C but none grew at

37 C as shown in Figure 1. The substances accumulated for each enzyme are fructose 1, 6

bisphosphate for 136, 3-phosphoglycerate for 152, phosphoenolpyruvate for 189, 2-

phosphoglycerate for 195, and pyruvate for 164, 166, and 211. In Figure 2, experiment 2 shows

that strains 136, 152, 189, and 195 grow on pyruvate while strains 164, 166, and 211 do not grow

on this medium. When put on fatty acids of a different molecular structure, all of the strains

tested(164, 166, and 211) are able to grow as shown in Figure 3. Mostly from the substances

accumulated, it can be inferred that the enzyme affected by a mutation is aldolase for 136,

phosphoglyceromutase for 152, pyruvate kinase for 189, enolase for 195, and pyruvate

dehydrogenase for 164, 166, and 211.

Discussion

From experiment 2, it can be concluded that strains 136, 152, 189, and 195 all must have

mutations in a gene for an enzyme used in glycolysis since they did not grow when given

glucose in experiment 1. If an enzyme is non-functional, then the substrate must accumulate.

From the Nuclear Magnetic Resonance (NMR) technique, it can also be concluded that the

mutation is in aldolase for strain 136 since fructose 1, 6 bisphosphate accumulates, is in pyruvate

kinase for strain 189 since phosphoenolpyruvate accumulates, is in phosphoglyceromutase for

strain 152 since 3-phosphoglycerate accumulates, and is in enolase for strain 195 since 2-

phosphoglycerate accumulates. For the other three mutant strains, the mutation can only be in the

enzymes after glycolysis since pyruvate accumulates. Experiment 3 helps narrow down the

enyme affected for these strains since they all grow on fatty acids. Fatty acids are directly used to

make acetyl CoA to be used in the Krebs cycle. The only enzyme between pyruvate and acetyl

CoA is pyruvate dehydrogenase, so this must be the mutated enzyme for strains 164, 166, and

211. Through genetic crossing, it appears that the mutation for strain 211 is in a different gene

than that of strains 164 and 166. These different mutations might lead to an identical phenotype

(pyruvate dehydogenase cannot work) because the enzyme pyruvate dehydrogenase consists of

more than one polypeptide subunit. The genetic information for these subunits are found on

different genes and they combine to form a quaternary structure that is the enzyme pyruvate

dehydrogenase. If a mutation is found on either gene that encodes for a polypeptide subunit, then

the resulting enzyme will not work.

Works Cited

"Glycolysis." Uic.edu. University of Illinois at Chicago, n.d. Web. 5 Nov. 2014.

Kimball, John W. "Cellular Respiration." Users.rcn.com. Users.rcn.com, 17 Apr. 2014. Web. 5

Nov. 2014.