Immobilized Metal Affinity Chromatography

Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect

Enzymes have an active site which has a complimentary base to a specific substrate, when these bind an enzyme-substrate complex is

enable the substrate to bind to the enzyme and form the enzyme substrate complex and

The results recorded in (table 30) and (figure 32) Indicated that Cu+2 activated the enzyme at 0.01M concentration by 1.2 fold and the activity gradually decrease by increasing the metal concentration to 0.1M with activity 51.29U/mg protein. Na+ ion activate the enzyme when added with 0.1M by

Macromolecules BCM 261 10/13/2014 Caroline Venter 13019865 Introduction Background Many of the molecules that are crucial in living organisms and systems are very large and are usually made up of macromolecules. Macromolecules are organic molecules with a large molecular mass and consist of repeating units called monomers. These repeating monomers are formed via condensation or dehydration reactions (loss of water or other small molecules in order to join two molecules) and usually each have a small molecular mass which contributes to the overall large molecular mass of macromolecules (Jenkins, Kratochvíl, Stepto, & Suter, 2009).

A clarified and physiologic buffered (pH 7 to 8) is added to the antibodies and purified ZA2GP to form a mixture that is added to the immobilized ligand.

Did cytochrome c or any other protein bind to the anion exchange column? (Explain the reasoning behind your answer).

Catalysts are substances that lower the activation energy needed for a particular chemical reaction (Moore and Vodopich, 75). On these enzymes exists a region known as the active site that is shaped specifically to bind to certain molecules, called substrates, which go through a chemical reaction. Factors that can influence this binding between enzyme and substrate, and therefor enzymatic activity, include pH, salt concentrations of surrounding solutions, and temperature. (Moore and Vodopich, 76).

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

Furthermore, enzymatic activity may be affected by two inhibitors, competitive inhibitors, that bind to the active site blocking the substrate from binding, and noncompetitive inhibitors, allosteric inhibitors that change the shape of the active site blocking the substrate from binding. However, an allosteric activator increases an enzyme’s activity by keeping the enzyme’s active configuration; and a cofactor (metal ion) or coenzyme (organic molecule), located in the active site of the enzymes and assists directly with the catalyst (Raven et.al.,

Campbell and Farrell define proteins as polymers of amino acids that have been covalently joined through peptide bonds to form amino acid chains (61). A short amino acid chain comprising of thirty amino acids forms a peptide, and a longer chain of amino acids forms a polypeptide or a protein. Each of the amino acids making up a protein, has a fundamental design that comprises of a central carbon or alpha carbon that is bonded to a hydrogen element, an amino grouping, a carboxyl grouping, and a unique side chain or the R-group (Campbell and Farrell 61).

Contrasting with tafamidis hydrogen bonding is absent at the base of the binding pocket like with AG10, but the Cl substituents are instead located in HBP 3, where hydrophobically the subunits are bridged. [4] This absence of additional

For the presence of substrates or inhibitors, the zinc active site has several characters for the substrates binding and activation. Firstly, carboxylate group of the Glu-72 becomes coordinated in a more nearly monodentate way. Secondly, there is a hydrophobic pocket near the active site where the aromatic ring in the C-terminal end of the substrate molecule can reside into the pocket help cleave the terminal peptide bond. In addition, the coordinated water molecule can form hydrogen bond with Glu-270 beside the active site. Also several Arg and Tyr residues are positioned in the active site allowing them to participate in substrate binding and activation.

The program generated the list of peptides and their charges for both wild type and mutant sequences. A complete list of peptides and their charges can be found in appendix 1 through 4. The charge of a peptide can also be calculated manually by looking at the charges of the ionzable amino acids or groups. The maximum charge of a peptide can also be is determined by the sum of charges of the ionizable groups, the N terminus, and the C terminus. The ionizable amino acids are Asp, His, Cys, Tyr, Lys, Arg, and Glu.

In order to study the binding mode of different inhibitors with McFabZ protein, docking calculation was performed using autodock and autogrid from ADT tools. These nine inhibitors biochanin A, genistein, juglone, epicatechin gallate, quercetin, daidzein, fistein, and myricetin have been docked into the active site of McFabZ. Table 4 shows the binding energy and binding constant calculated by ADT tools.