Purpose of the Tutorial

Throughout this tutorial we will be targeting basic chemistry topics. A scientific research article will be used to demonstrate key chemistry topics we will be discussing. These topics are vital to the understanding of more advanced chemistry. There are interactive molecules incorporated into the text to help your understanding.

The summary of the research artical will be confusing, you are not expected to understand it completely to benefit from the tutorial. The reasearch artical is used as a refrence to demonstrate the chemistry topics we will be discussing.

Summary: Scientific Research Artical

The study where this molecule was obtained is named "Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis-Complex with Coenzyme A and Tobramycin". The study focused on AAC (2’)- Ic also known as aminoglycoside 2’- N- acetyltransferase. The scientist’s in the study determined the crystal structure of AAC (2’)-Ic from Mycobacterium tuberculosis. The specific fold of AAC (2’)-Ic places in the GNAT or GCN5-related N-acetyltransferase superfamily. Although the physiological function of AAC(2’)-Ic in not certain, the crystal structure they determined allowed them to hypothesize. Through the crystal structure they determined that this enzyme might acetylate mycothiol. Mycothiol is key biosynthetic intermediate and the major reducing agent in mycobacterium. This enzyme is capable of acetylating aminoglycosides bearing a 2’ amino group, when this occurs the aminoglycoside antibiotic becomes inactive.

Objectives

By the end of this tutorial you should be able to:

1. Describe and provide examples of covalent bonds, ionic bonds, and hydrogen bonds

2. Understand and identify the importance of secondary structures

3. Understand and describe an active site

4. Describe what a ligand is

5. Understand and explain the importance of Tobramycin as an antibiotic

Types of Bonds

There are 3 common types of bonds. A hydrogen bond, covalent bonds, or an ionic bond. The strongest bond is a covalent bond followed by the ionic bond, leaving the weakest bond to be the hydrogen bond. Covalent bonds, the strongest type of bond, they involves the sharing of electrons between two molecules. An example of a covalent bond is hydrochloric acid or HCl. The electrons are being shared between the chlorine atom (Cl) and the hydrogen atom (H). An ionic bond is an attraction between two molecules of opposite charge. The opposite charges I am referring to are a positive (+) and a negative charge (-). A positively charged atom is referred to as a cation, and a negatively charged atom is referred to as an anion. Hydrogen Bonds, the weakest of bonds, are attractive interactions (dipole-dipole) between an electronegative atom and hydrogen. Electronegative atoms are atoms that have high electron density. They are strong atoms that pull electrons towards then from weaker/low electron density atoms, such as hydrogen. When the electronegative atom pulls the electrons it leaves the other atom with a slight positive charge. The most common example of hydrogen bonding is water. The water molecule chemical formula is H2O. The highly electronegative oxygen pulls the hydrogen closer by attracting hydrogen’s electrons allowing the formation of a water droplet. The electronegative atoms allow for the droplet to be held together instead of spreading. The hydrogen bonds in this picture are displayed as yellow dashed lines. The hydrogen bonds in this molecule are important to the secondary structures providing the stability of the atoms orientation.

Secondary Structures

Secondary structures are alpha helices and beta sheets. They help contribute to the stability of the molecule. The alpha helices are represented with pink arrows and the beta strands are represented with yellow arrows. This molecule has approximately four alpha helices and two beta strands, when presented as a monomer. Since this structure is represented as a dimer you actually have eight alpha helices and four beta sheets. The concept of a dimer is explained in the "Ligands" section later on in the tutorial. Alpha helices rotate in a clockwise manner and are also oriented in a parallel formation. The parallel alpha helices are held together by hydrogen bond, which we discussed earlier. Beta sheets are often anti-parallel. The structure of the alpha and beta sheets in Tuberculosis/CoA/Tobramycin structure represents the GNAT fold. The folding of a protein is what gives the function. When you have a change in the folding you have a change in the function. The GNAT fold described in the study has a function of acetylation. Acetylation is the addition of an acyl group. The chemical formula of an acetyl group is COCH3. It is important to note that the discovery of the GNAT fold lead to the understanding of the major function.

Active Site

The active site of a molecule can be described as a pocket where an interaction between substrates causes a physiological effect by causing a change in conformation. The conformation is referring to the orientation of the molecules involved in the structure. The conformation change can inhibit or activate the physiological effect. The active site is where the ligand is going to bind. (Ligands are discussed in detail later on in the “Ligands” section) The active site can either be inhibited or activated by ligands. Referring back to our article, the active site is where the substrate, in this case tobramycin, binds to CoA and the mycobacterium to cause an antibacterial effect. It the study described this is where the acetylation of the tobramycin should be occurring. The acetylation of tobramycin would cause the tobramycin to be inactive, hence inhibit the active site.

Ligand

Ligands are molecules or complexes that are within the secondary structures that orient in such a way to contribute the function of the complex as a whole. Ligands can have binding sites on receptors, and when bound can trigger a physiological response. A ligand can be a competitive agonist, allosteric agonist, competitive antagonist, or an allosteric antagonist. An agonist is a ligand that causes a physiological response, activating the active site. An antagonist is a ligand that inhibits a physiological response, not allowing the active site to be activated. When a ligand is competitive that means that the ligand is binding to the same site as the physiological activator, hence it is competing for the same site. When a ligand binds to an allosteric site, the ligand is binding to the same receptor but it is not binding to the active site. The ligands present in the complex used by the research article are coenzyme A, Tobramycin and Phosphate-Adenosine-5'-Diphosphate.

Coenzyme A

Coenzyme (CoA) is a coenzyme that synthesizes and oxidizes fatty acids. This process is essential for the utilization of fatty acids. Coenzyme A is used as a substrate in the citric acid cycle. The citric acid cycle is also known as the Krebs cycle or tricarboxylic acid cycle (TCA). This process is important to the production of ATP. ATP is an energy source used by the body. PAP is not mentioned in this tutorial because it is not a commonly used enzyme. The Protein’s in this molecule are represented as a dimer. A dimer is a chemical structure formed from two subunits. These subunits are identical. Some molecules are present as a dimer because it is more stable then the monomer. The dimer is constructed by connecting two subunits along their axis.

Tobramycin

Tobramycin is an antibiotic part of the aminoglycoside family. Aminoglycosides produce antibacterial effects by inhibiting protein synthesis and compromising the cell wall structure. By inhibiting the protein synthesis of the bacteria it does not allow the bacteria to replicate. The cell wall is an important structure to bacteria, because it provides the structure and stability to the bacteria. By disrupting the cell wall we are removing the stability of the bacteria and ultimately casing bacteria death. Tobramycin targets a variety of bacteria particularly gram(-) species. Just like all drugs there are side effects associated with tobramycin. Some of the more common side effects are ototoxicity and nephrotoxicity. Ototoxic is hearing loss and nephrotoxic is causing kidney damage. The kidney damage is due to Tobramycin reabsorption through the renal tubules. This basically means that tobramycin may be toxic to the kidneys and the toxicity is caused by the contact-time in the renal tubules where the drug is located. Tobramycin trade name is Tobrex. A trade name is another name for tobramycin. It is a pregnancy category D. Pregnancy categories are assigned to all drugs. They are used to classify how likely the drug is to cause harm to the fetus. The pregnancy categories are A, B, C, D, and X. Pregnancy category A causes no harm to the fetus and pregnancy category X, which indefinitely causes harm to the fetus. Since Tobramycin is a pregnancy category D, this is not an optimal choice for a pregnant patient. Tobramycin can be given intravenously, intramuscularly, as an inhalation or ophthalmicly. Intravenously is an IV route of administration where the drug is administered directly to the vasculature or blood vessels. Intramuscular is a shot that penetrates your muscle. A common example of an intramuscular administration would be a flu shot. Inhalation is a route of administration where the lungs are the targets. An example of this would be an inhaler used in asthmatics. Ophthalmic administration is where the drug is administered to the eye; an example would be an eye drop.

This molecule displays the protein bound to the ligand CoA. The red molecules represent an anionic or negatively charged interaction. The dark blue molecules emphasize the cationic or positively charged interactions. The cationic and anionic interactions are contributed to arginine, aspartic acid, or glycine amino acids. The light blue molecules represent histidine, which is a basic amino acid. The difference in the charges displayed here contribute to the stability of the molecule. Since the charges are different it allows the molecules to be attracted to the opposite charge holding the molecule in a stable position.

Amino Acids

Amino acids are the building blocks of proteins. There are 20 common amino acids. The contain and amine group (-NH2), a carboxylic acid group (-COOH) and a functional group specific to each amino acid. The functional group determines how the amino acid is classified. They are categorized as either, polar, non-polar, acidic or basic. There are 8 different amino acids present in the . CoA has a combination of 7 amino acids bound to it. The amino acids are two Arginine (basic amino acid), one Glycine (polar amino acid), and four Valine (non-polar amino acid). PAP has four amino acids bound to it, two Histidine and two tryptophan (non-polar amino acid). Tobramycin also has four amino acids bound to it, two aspartic acid (acidic amino acid)), Serine (polar amino acid) and tryptophan (non-polar amino acid)

CoA Amino Acids:

In this representation it displays the covalent bond between CoA and Arginine 124. Arginine is displayed as the pink molecule and CoA is displayed as the orange and red molecule. Arginine classified as a basic amino acid and is a nonessential alpha amino acid, meaning that can be synthesized by the human body.

Val96 and CoA are bound by a hydrogen bond, clearly displayed in this representation.

PAP Amino Acids:

This shows His54 bound to bound through a hydrogen bond.

The other Amino acid bound to PAP with a hydrogen bond is tcp

• References:

<Vetting, M. W., et al. "Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis-Complex with Coenzyme A and Tobramycin." RCSB Protien DataBase. N.p., 28 Aug.2002. Web. 13 July 2011. <http://www.rcsb.org/pdb/explore/explore.do?structureId=1M4D>.>