172 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Figure 6-21. Cells process many substrates into ‘products’ through biosynthesis. In simple reactions, a substrate is turned into a
product. In complex pathways, the products of one enzyme reaction become the substrate of another enzyme reaction. Image Copy-
right Kyoto Encyclopedia of Genes and Genomes (https://www.genome.jp/kegg/)
Notice that some of the open circles in Figure 6-21
don’t have names to them yet. However, once you
complete the Exploring Metabolic Pathways Online
Websearch Breakout, you will be able to explore the
biosynthesis pathways online.
In Table 6-2 is the step by step metabolic process for
converting the sugar D-fructose to the amino acid
L-histidine. Each line of the table can be read as:
“In step ___ of the histidine amino acid biosynthe-
sis pathway, the substrate molecule _________ is
converted to the product molecule _________ by the
protein enzyme _________”.
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173Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Exploring metabolic pathways online Web Search Breakout
Exploring metabolic pathways online: Biosynthesis maps are an essential part of engineering cells to
produce molecules for us. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a great resource
to nd molecules that are made biologically, to understand the chemical reactions in cells that result in
the production of the molecules, and to ultimately discover what DNA sequence(s) in the cells blueprints
encode for the proteins that cause the chemical reactions. Try searching “Kegg fructose amino acid synthe-
sis” on a search engine to nd the biosynthesis map 01230 in Figure 6-21.
Table 6-2. Non-coding regions that function in DNA and RNA
Step # Starting molecule (substrate) End molecule (product)
Protein enzyme catalyzing the reac-
tion
1 D-fructose D-fructose-6P hexokinase
2 D-fructose-6P D-arabino-3-Hexulose 6P 6-phospho-3-hexuloisomerase
3 D-arabino-3-Hexulose 6P D-Ribulose 5P 3-hexulose-6P synthase
4 D-Ribulose 5P D-Ribose 5P ribose-5P-isomerase A
5 D-Ribose 5P 5-Phosphoribosyl diphosphate ribose-phosphate pyrophosphokinase
6 5-Phosphoribosyl diphosphate 1-(5-Phosphoribosyl)-ATP ATP phosphoribosyltransferase
7 1-(5-Phosphoribosyl)-ATP 1-(5-Phosphoribosyl)-AMP
phosphoribosyl-ATP pyrophosphohy-
drolase
8 1-(5-Phosphoribosyl)-AMP
Phosphoribosyl-formimino-AI-
CAR-phosphate
phosphoribosyl-AMP cyclohydrolase
9
Phosphoribosyl-formimino-AI-
CAR-phosphate
Phosphoribulosyl-formimino-AI-
CAR-phosphate
phosphoribosylformimino-5-ami-
noimidazole carboxamide ribotide
isomerase
10
Phosphoribulosyl-formimino-AI-
CAR-phosphate
D-erythro-Imidazole-glycerol 3P cyclase
11 D-erythro-Imidazole-glycerol 3P Imidazole-acetol phosphate
imidazoleglycerol-phosphate dehy-
dratase
12 Imidazole-acetol phosphate L-Histidinol phosphate
histidinol-phosphate aminotransfer-
ase
13 L-Histidinol phosphate L-Histidinol
imidazoleglycerol-phosphate dehy-
dratase
14 L-Histidinol L-Histidine histidinol dehydrogenase
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174 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Summary and What’s Next?
Congratulations, in this chapter you dug deep into
the world of chemistry and learned protein enzymes
catalyze chemical reactions by reducing the amount
of energy required to make or break bonds resulting in
substrates being processed into products. This is what
we call Enzymatic Processing, which is the third step
and nal step of the Three Steps to Microfacturing.
You learned about how an atom is made up of a dense
positively charged nucleus and electron clouds called
orbitals around the nucleus. The electrons in the outer
valence orbitals can overlap with those of other atoms
to create covalent bonds. You also learned about how
electromagnetic bonding is present at different levels
in ionic bonding, hydrogen bonding, and even Van der
Waals interactions and London Dispersion Forces. You
saw that while covalent bonding is critical in holding
atoms together to form molecules, hydrogen bonding
is critical to hold molecules together loosely. Hydrogen
bonding is key for holding DNA strands together, help-
ing proteins interact with DNA, for substrates binding
to enzymes, and a lot more.
Lastly, we looked into an actual reaction mechanism in
which an enzyme catalyzes changes in molecules. CAT
binds acetyl-CoA and chloramphenicol in a reaction in
which CAT initiates a chain of electron jumping events
that causes an acetate group to be transferred from
the acetyl-CoA to the chloramphenicol rendering the
antibiotic inert and unable to harm the cells.
These Fundamentals were reinforced by the hands-on
exercises you completed where you completed enzy-
matic reactions involving one or more substrates. In
those reactions, you processed small molecules to
change odor, change color and even create oxygen.
You’ve only touched the surface of the world of chem-
istry and bonding, and as you continue your journey
into genetic engineering beyond this book, you will
be learning more about this subject. Mastering the
concepts of chemistry and bonding are essential to
mastering your abilities to manipulate cells through
genetic engineering.
In the next chapter, you are going to look deeper into
genetic regulation. Up until now the DNA plasmids
you have used were completely autonomous, meaning
once they were in the cells, they did everything auto-
matically. In the next chapter, you’re going to learn
how you can turn genes on and off using different
mediums such as chemicals, temperature, and light.
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175Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Review Questions
Hands-on Exercise
1. Why does one waft?
2. What is the difference between the two negative control plates in the Smell-it Experiment?
3. What are the experiment steps to get you to microfacture banana smell molecules?
4. What made it feasible to complete the Blue-it Kit using cell extract rather than in the cells (like the
Smell-it Kit)
5. What is DTT? Why is it used in the lysis and reaction buffers?
Fundamentals
1. Explain the Four B’s in relation to a one or two substrate-enzyme reaction.
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176 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
2. How is the classic illustration of an atom incorrect? (Figure 6-10)
3. What is a mole?
4. Describe how a single electron can create an orbital.
5. What shapes do s-orbitals and p-orbitals have?
6. What are the four major types of bonding?
7. What type of bonding is used when two strands of DNA zip together to form the double helix? Explain.
8. How does chloramphenicol acetyltransferase start the chemical reaction that is important for selection
during genetic engineering?
9. What is the difference between a simple enzyme reaction and a complex enzyme reaction?
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