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. Describe Primary Structure

3. Understand and identify the importance of secondary structures

4. Understand and describe an active site

4. Describe what a ligand does. Competitive/Allosteric inhibitors and inducers

7. Be able to classify amino acids and understand what the classification represents

The "element color code" drop down box maybe needed throughout the tutorial for identification of atoms.

Periodic table

^[3]

An atom is the smallest unit of matter. Protons, neutron, and electrons, make up the composition of an atom providing its physical features. The common physical features of atoms are used to organize them in the periodic table. Electrons are negatively charged particles that surround the atom’s nucleolus. Protons are positively charged particles and neutrons are uncharged. Both neutrons and protons are located in the central part of the atom known as the nucleus.

The image to the left displays Oxygen (O) in its periodic box. The atomic mass of oxygen is 8, centered above the single-letter abbreviation of Oxygen. “8” represents the number of protons in the atom’s nucleolus. The atomic number is also used to arrange elements in the periodic table, notice the increasing value of protons in the atoms from left to right. The atomic mass of an element is centered under the single-letter abbreviation of the atom and represents the total weight of the atom, in this case oxygen has an atomic mass of ~16. The total weight of an atom is the sum of the atoms protons, neutrons and electrons.

The periodic table is a collaboration of atoms that are arranged according to their common features. Groups are the vertical columns of atoms, and the periods are the horizontal columns. When moving from left to right across the periods (horizontally), there is an increase in electronegativity. Electronegativity refers to the atoms ability to pull other electrons towards it, increasing its density and providing it’s self with a negative charge. The concept of electronegativity contributes to the polarity of a molecule.

Polarity is caused by a difference in electronegativity between atoms in a compound and/or the asymmetry of the compounds structure. A compound is nonpolar when the electronegativity of the atoms/functional groups are close or even. The compound is symmetrical and the electrons are not being pulled more in one direction.

Amino acids

Amino acids are the building blocks of proteins. There are about 500 amino acids but we will be referencing the 20 more common ones. Polymer chains (peptides) join amino acids together to form proteins. The process of protein formation is known as translation. During translation, a complex known as the ribosome is responsible for adding amino acids to a peptide chain to form a complete protein.

20 More Common Animo Acids

The basic structure of an amino acid is an amine group (-NH2), a carboxylic acid (-COOH), hydrogen (H), and a functional group (R). The functional group is what varies between amino acids. The functional group determines how the amino acid is categorized. They are categorized as essential/nonessential, polar/non-polar, and acidic/basic.

Classification of Amino Acids

Essential vs. Nonessentail Amino Aicds

An amino acid is considered essential when the human body is not capable of synthesizing/producing it. These amino acids need to be obtained from our diet. If the body is capable of producing an amino acid through metabolism or another method, the amino acid is classified as nonessential.

Neutral, Polar vs. Nonpolar Amino Acids

The polarity of an amino acid is dependent on the difference in electronegativity and the asymmetry of the compounds structure, discussed previously. For example, Arginine is a polar amino acid and Glycine is a nonpolar amino acid. As we learned earlier, an amino acid structure consists of a carboxylic acid, an amine, hydrogen, and a functional group. Look at the structure of arginine; the amine group, carboxylic acid and hydrogen are located towards the bottom of the representation. The functional group is the large extension of atoms upward. You can see from this image that the functional group has a greater density/electronegativity compared to the rest of the structure, hence making this amino acid polar. In contrast the structure of glycine, located next to arginine, has little polarity. The functional group attached is only a methyl group (CH3), which has low density/electronegativity compared to the rest of the structure making glycine a nonpolar amino acid. Neutral amino acids have functional groups that are similar in electronegativity, so the electrons are not pulled in one direction more dominantly then another. In other words, there is no increase in density to one side. When an amino acid is neutral, it is less reactive then a polar amino acid. It is less reactive because the structure is stable. A polar amino acid is pulling electrons yielding a slight positive and negative charge with in the amino acid structure, making it less stable. Those charges want to react with other atoms, yielding the higher reactivity.

Neutral Amino Acids

• Alanine (ala)
• Asparagine (asn)
• Cysteine (cyc)
• Glutamine (gln)
• Glycine (gly)
• Isoleucine (ile)
• Leucine (leu)
• Methionine (met)
• Phenylalanine (phe)
• Proline (pro)
• Serine (ser)
• Threonine (thr)
• Tryptophan (trp)
• Tyrosine (tyr)
• Valine (val)

Acidic vs. Basic Amino Acids

An Example of a pH scale is located above. A pH scale is a scale that ranges from 1-14. An acidic pH is a pH below 7, a basic pH is greater then 7, and a pH of 7 is considered neutral (ex: water). Some common acidic functional groups are alcohols (OH) and carboxylic acids (COOH). Some examples of basic functional groups are amines (NH3) and ethers (CH2OCH2). You can predict if an amino acid will be acidic or basic according to its structure. For example, glutamic acid contains a carboxylic acid functional group, which we stated previously to be acidic and lower the pH of the amino acid. On the other hand, arginine is classified as a basic amino acid. The functional group of arginine contains a guanidine group. A guanidine group contains amino groups, increasing the pH of the amino acid.

Types of Bonds

There are many different types of bonds that occur physiologically. Ionic, covalent, and hydrogen bonds are some of the most abundantly seen. The strongest of these is the covalent bond, followed by the ionic bond, which leaves the weakest bond to be the hydrogen bond. Knowing where these bonds are utilized will aid in your understanding of why a structure is in a certain conformation.

Covalent Bonds

Covalent bonding

The strongest of these bonds is the covalent bond. Covalent bonds involve sharing pairs of electrons between two atoms. These bonds are very stable. Figure two, shown to the left represents Coenzyme A (CoA) with the addition of an acyl group to the sulfur atom. The introduction mentioned that the addition of the acetyl group is important to the study because when it is transferred to Tobramycin, the antibiotic becomes inactive. The solid connections between the atoms are representation of the covalent bonds.

Ionic Bonds

Ionic Bonding^[5]

An ionic bond is when an electron(s) is transferred from one atom to another due to the opposite charges of the atoms. The positively charged cation is attracted to the negatively charged anion. In the image to the right, you see an anion, Fluorine (F) and the cation, Sodium (Na). The double-sided arrow between them is representation of their attractive force. Fluorine has a higher electronegativity than sodium. As we discussed previously, when an atom has higher electronegativity it pulls electrons from the lower electronegative atom, in this case sodium. The transfer of the sodium electron (blue) is shown using an arrow. This representation highlights an ionic interaction between Tobramycin and Aspartic acid (Asp). The nitrogen on Tobramycin has a (+) positive charge and Aspartic acid has a (-) negative charge. The opposing charges are attracted to each other forming an ionic bond, holding the compounds in close proximity.

Hydrogen Bonds

Hydrogen Bonding^[6]

The weakest bond discussed here, the hydrogen bond is an attractive interaction between an electronegative atom and hydrogen. When the electronegative atom pulls the electrons, it leaves the other atom with a slightly positive charge. A common example of this is water. The image to the right demonstrates the hydrogen bonding in water. The highly electronegative oxygen pulls the hydrogen closer by attracting hydrogen’s electrons. When oxygen pulls the electrons, it leaves hydrogen with a slight positive charge. Since oxygen is pulling the hydrogen’s inward, the formation of a water droplet is possible. In this representation the hydrogen bonds are represented as yellow-dashed lines. The hydrogen bonds are important to this compound in the study because they offer stability to the secondary structures.

Primary Structure

The primary sequence of a protein is the amino acid linear sequence of the compound. For example the linear sequence of the protein enzyme AAC (2’)-Ic is shown below.

The letters in the primary structure are the single-letter abbreviations of the amino acids. The single-letter abbreviations are given in the amino acid portion of this tutorial. The primary sequence of AAC (2’)-Ic, a protein enzyme, was an important discovery to the study because it revealed the cause of the Tobramycin acetylation, leading to its inactivity.

Secondary Structures

Secondary structures are alpha helices and beta sheets. The helices and sheets provide stability to the compound as a whole. The alpha helices are represented with pink arrows and the beta strands are represented with yellow arrows. This molecule has approximately eight alpha helices and four beta sheets. Alpha helices have a cylinder-like structure with a parallel formation. This representation shows these key features of alpha helices. Here, you can see the parallel formation within the cylinder structure. The parallel alpha helices are held in its cylinder structure by hydrogen bonds. Beta sheets are often anti-parallel, which can be seen in this representation. The folding of a protein, alpha helices and beta sheets, are what gives the compound its function. When there is a change in protein folding, the function will change. As previously stated, the study discovered ACC(2’)-Ic to have a GNAT fold, and the GNAT family are a group of enzymes capable of acetylation.

Active Site

The active site of a molecule can be described as a pocket where an interaction between compounds occurs. This interaction will cause a change in structure shape. The conformational change, change in structure shape, can inhibit or activate the physiological/pathological affect. The active site can either be inhibited or activated by the compound that binds there. Referring back to our article, the active site is where the acetylation is going to occur. In this depiction of the active site you can see the pocket where Coenzyme A (CoA) will aid the enzyme in the acetylation of Tobramycin (Toy), the aminoglycoside antibiotic. The enzyme is the "blob" surrounding the antibiotic and CoA. The enzyme/protein is holding these compounds in a conformation forcing them to react. The acetylation at the active site will cause the antibiotic to be inactive, hence inhibiting the active site. When Tobramycin becomes inactivated it is no longer able to aid in the destruction of bacteria. This is what we call antibiotic resistance.

Substrates

A substrate is a compound that is acted upon by the enzyme. The substrate binds the active site of the enzyme, once bound the enzyme transfers a functional group(s) to the substrate. After the substrate has collected the functional group(s) it is released from the active site. When this occurs the substrate can go on to bind the enzymes compound of interest and transfer the acquired functional groups to produce the product. Once the final transformation occurs the product is either inhibited or activated by the conformational change.^[3]

This equation is a representation of the interaction between a substrate and an enzyme. As you can see the enzyme (E) and substrate (S) start off as two stand alone compounds, but then join together at the enzymes active site to form the enzyme-substrate (ES) compound. Once the enzyme releases the substrate you are left with the final product (P) and the enzyme (E).

Acetylation Reaction ^[2]

From the article summary you know that AAC(2’) has a similar fold to that of the GNAT superfamily. The GNAT fold described in the study has the function of acetylation, the addition of an acetyl group. An acetyl functional group is composed of CH3CO. It is important to note that the discovery of the GNAT fold lead to the understanding of the function of AAC(2’). The reaction centered above is the acetylation that occurs to the aminoglycoside antibiotic causing its inactivity. The Acetylation was reconstructed and modified from the article “Aminoglycoside 2’ –N- Acetyltransferase from Mycobacterium tuberculosis in complex with Coenzyme A and aminoglycoside substrate”, the research article we have been referencing. From the reaction centered above you see the aminoglycoside antibiotic (Ribostamycin) being acted upon by the enzyme AAC(2’). AAC(2’) is adding and acetyl group to the antibiotic using the substrate CoA. On the right side of the arrow you can see the final product of the acetylation, the antibiotic and acyl group bound. The Acetyl group is circled, so you are able to locate it throughout the reaction. Acetylation is one of the more common reactions that occurs.^[2]

Ligand

Ligands are molecules or complexes that are located within the secondary structures. The ligands are held in a certain conformation by the secondary structure. This conformation contributes to the function of the compound, as mentioned before. Ligands can have binding sites on receptors. When bound to the receptor it 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. A ligand is competitive when it 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 research article complex are coenzyme A, Tobramycin and Phosphate-Adenosine-5'-Diphosphate. The ligands in this complex are represented as a dimer. A dimer is a chemical structure formed from two identical subunits. Some molecules are present as a dimer because it is more stable then the monomer, one subunit. The dimer is constructed by connecting two subunits along their axis.

The hide-aways boxes located below provide more information about CoA and Tobramycin for those who are interested.

• References:

http://www.elmhurst.edu/~chm/vchembook/561aminostructure.html