Exam 3: Enzymes and Therapeutics

How is the substrate concentration and the reaction velocity related to each other on a graph?

As the substrate concentration is on the X axis, the Vmax is the utmost value it can reach. Where half of the Vmax is on the y axis, that intersecting point is the Km

What is the role of chymotrypsin?

Chymotrypsin cleaves the peptide bond after an aromatic amino acid

How do pancreatic enzymes function?

Trypsinogen is excreted from the pancreas, and functions with enterokinase from small intestine. Trypsin is released from the small intestine, while at the same time other inactive proteases from the pancreas are released. Active forms of proteases take role in the small intestine, ultimately leading to protein digestion in the small intestine.

How is chymotrypsin produced?

It is cleaved by trypsin

What is the specific biochemical process that allows chymotrypsin to become active?

Chymotrypsin starts out as inactive in the form of chymotrypsinogen, in a 245 nucleotide long strand. Trypsin cleaves chymotrypsinogen to make active pie-chimotrypsin, producing arginine 15 and isoleucine 16 in the process. pie chimotrypsin undergoes autolysis, producing serine 14, arginine 15, threonine 147 and asn 148. The 245 long nucleotide strand now has breaks at 13-leucine, 16-Ile, 146 tyrosine, and 149 alanine alongside active alpha chymotrypsin.

What enzymes are involved in the catalytic triad?

ASH (aspartic acid, serine, histidine)

or, DSH if using the single letter acronym (in respective order)

The first step of the chymotrypsin mechanism is substrate binding; what occurs in this step?

As a substrate binds, the side chain of the residue adjacent to the peptide bone to be cleaved nestles in hydrophobic pocket on enzyme. Peptide bond is positioned for attack

The second step of the chymotrypsin mechanism is the nucleophilic attack; what occurs here?

A strong, nucleophilic alkoxide ion is made as serine 195 and histamine 57 interact. The ion attack the carbonyl group from the peptide, forming a tetradehral acyl-enzyme. The carbonyl oxygen of the substrate in turn experiences the formation of a short lived negative charge, which is stabilized by hydrogen bonding in the oxyanion hole.

*The short lived intermediate is termed acylation

After acylation in the chymotrypsin mechanism, what is the third step?

Since the substrate carbonyl oxygen carries a negative charge, the formation is unstable. As the tetrahedral intermediate collapses, carbon reforms a double bond. The bond holding the carbon and the amino group of the peptide linkage is broken, displacing the peptide bond. Amino leaving group is protonated by histadine-57.

In the fourth step of the chymotrypsin mechanism, water comes in. What happens?

An incoming water molecule is deprotonated by general base catalysis, generating a strongly nucleophilic hydroxide ion. The ester linkage of the acyl-enzyme has a hydroxide ion that is attacked, generating a second tetrahedral intermediate where the oxygen in the oxyanion hole has a negative charge.

The fifth step of the chymotrypsin mechanism is a water attack; what occurs here?

The acyl-enzyme intermediate will become short lived, undergoing deacylation. The oxygen of the added water molecule will form a bond with the carbonyl carbon, while the double bond between the carbon and oxygen produces an oxyanion. Carbonyl carbon now has an OH group attached to it. This results in deacylation.

The sixth step in chymotrypsin mechanism is deacylation collapse. What occurs here?

The tetrahedral intermediate collapses again, forming carboxylate anion and displacing serine-195.

The seventh step of the chymotrypsin mechanism is dissociation of the second product. What occurs here?

Free enzyme is regenerated as second product dissociates from the active site.

Who discovered DNA polymerase 1?

Arthur Kolnberg

What are the four components of DNA polymerase 1?

Finger, thumb, palm, and the area of exonuclease activity

What are the four roles that DNA polymerase I is responsible for?

Both RNA independent and RNA dependent DNA polymerase activity in the 5’ to 3’ direction; and exonuclease activity in both directions

What are Klenow fragments?

Klenow fragments are large sections of DNA polymerase I that are products of E.coli and DNA polymerase I interactions. Klenow fragments retain polymerization and 3’ to 5’ exonuclease activity, but have lost exonuclease activity in the 5’ to 3’ direction.

Which termini is not degraded in the Klenow fragments?

The 5’ termini is not degraded

What is exonuclease activity responsible for?

Exonucleases remove the RNA primer at the initiation point of replication; primase is responsible for creating the RNA primer

DNA polymerase I is also known as the “holoenzyme”; what three domains constitute the structure of the holoenzyme?

The 3’ to 5’ exonuclease domain is bound in between the polymerase domain and the 5’ to 3’ exonuclease domain

During the formation of the Klenow fragments, what components of the holoenzyme remain together and/or are separated?

The Klenow fragment consists of the polymerase domain, and the 3’ to 5’ exonuclease domain. The 5’ to 3’ exonuclease domain is separated

What is the function of DNA polymerase I?

DNA polymerase 1 checks the geometry and alignment between the four nucleotides found in DNA

When does DNA polymerase 1 fall off?

It falls off from the replication fork after adding 50 to 60 nucleotides

How does CRISPR Cas 9 work?

As an RNA mediated DNA recognition system, it is utilized by bacteria in which a short RNA sequence recognizes a viral DNA sequence. As the DNA and RNA sequence form a complex, Cas9 enzyme cleaves the DNA

The RNA sequence that is used to recognize the DNA sequence is known as guide RNA; how long can this guide RNA be?

Between 19 to 20 nucleotides

In the CRISPR Cas 9 system, how does the bacterial DNA recognize the viral DNA?

Viral DNA contains “flags” that are protospacer adjacent motifs.

What are the two different kinds of PAM found?

With both PAM’s, they are both recognized in the 5’ to 3’ sequence. Streptococcus pyrogenic recognises NGG; S. aureus recognizes NNGRR

How does DNA repair via base excision repair work?

When a nucleotide cannot be directly repaired and instead must be replaced, DNA glycosylases cleave the glycosidic bond of the altered nucleotide, leaving behind a deoxyribose residue with no attached base. An AP endonuclease will cleave one side of the deoxyribose residue, producing deoxyribose and several adjacent residues that are removed by cellular exonuclease. The remaining gap is sealed and filled in with DNA polymerase and DNA Ligase.

In other cases where the glycosidic bond is hydrolyzed, alternative apurinic/apurimidic/abasic sites can result.

When pyrimidine dimers, displaced bases, and bases with bulky substituents are present, what biochemical process resolves this issue?

Nucleotide excision repair recognizes helical distortions instead of a specific sequence; nucleotide excision repair is the common method to battle sunlight and tobacco smoke as carcinogens.

How does nucleotide excision repair work?

As an ATP-dependent process, there are proteins UvrA, UvrB,UvrC, and UvrD. At the seventh and third/fourth phosphodiester measured from the 5’ to 3’ side, the 11 to 12 long nucleotide sequence is excised through UvrD (a.k.a. helicase II).Polymerase I and DNA Ligase come in to complete the job.

What is the detailed redundant system for when nucleotide excision repair works?

MutS will bind prior to MutL binding. Once MutL binds, ATP disintegrates into ADP and an inorganic phosphate molecule. A loop forms prior to MutH binding, where UvrD excises. DNA Polymerase 1 and DNA ligase follow

How does cloning DNA work?

First, a restriction enzyme, PCR, or chemical synthesis will create a fragment of DNA of appropriate size. Then, the fragment is incorporated into the vector which contains necessary sequences to induce DNA replication. Now, the vector containing the DNA of interest is incorporated into cells, and replicated before being selected.

What does the origin of replication do on a plasmid?

Recruits replication machinery proteins to allow initiation of replication; is a DNA sequence itself

What does the antibiotic resistant gene on a plasmid do?

Allows for the selection of plasmid containing bacteria

What do the multiple cloning sites on a plasmid do?

These short fragments of DNA contains several restriction sites that allow for the easy insertion of DNA. The multiple cloning sites are located downstream from the promoter

What does the insert in a promoter do?

The insert is a gene, promoter, or other DNA fragment cloned into the multiple cloning sites for studies.

What does the promoter region in a plasmid do?

Promotes transcription of the target gene; essential for expression vectors. Determines in which cells the gene is expressed and amount of recombinant protein obtained

What does the selectable marker do?

Antibiotic resistance gene allows for selection in bacteria; but in other plasmids, have selectable markers for use in other cell types

What does the primer binding sites do?

The primer binding sites is an invitation point for PCR amplification or sequencing. It is a short single stranded DNA sequence that can be exploited for sequence verification of plasmids

What do the genes amp, lacZ, and lacL corresspond to?

Amp corresponds to antibiotic resistance of ampicillin; lacZ is responsible for the enzyme Beta-galactosidase, and lacL corressponds to a factor that encodes the transcription of lacZ

The most common way to study gene expression is through transformation; how does transfection vary with this?

Once the plasmid has been created, it is inserted into a mammalian cell. The mammalian cell will then excrete either secretory or membrane-bound proteins. If it is secretory, it will go into media. If it is membrane bound, it will go to the media once the membrane targeting sequence is removed.