What are Enzymes?
Enzymes are biological polymers that catalyze biochemical reactions. The vast majority of enzymes are proteins; however, there are some catalytic RNA molecules called ribozymes.
Enzymes, the biological catalysts are highly specific, catalyzing a single chemical reaction or a very few closely related reactions. The exact structure of an enzyme and its active site determines the specificity of the enzyme. Substrate molecules bind themselves at the enzyme’s active site. Substrates initially bind to the enzymes by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Enzymes lower the activation energy and the reactions proceed toward equilibrium more rapidly than the uncatalyzed reactions. Both prokaryotic and eukaryotic cells commonly use allosteric regulation in responding to changes in conditions within the cells.
Allosteric regulation can be positive or negative. Regulation by allosteric inhibitors is common in many biosynthetic pathways. A protective peptide in zymogens regulates by inactivating the protein. Zymogens are proteolytically activated. The catalytic activity of some enzymes may be regulated by cofactors. Metal ions or other small molecules serve as the cofactors. Some enzymes contain tightly bound cofactors termed prosthetic groups. Protein degradation is also one central way to regulate the enzyme levels. Hundreds of enzymes are commercially available. Some of these have increasing importance in industry and in medical and clinical applications.
Features of Enzymes
- An enzyme’s function is intrinsically linked to its three-dimensional structure, determining how it performs substrate binding, catalysis, and regulation. X-ray crystallography has been the most important technique in the development of our understanding of enzyme structure and hence enzyme function. Nuclear magnetic resonance (NMR) has also been used successfully to study many structures, but crystallography remains the principal technique for structure elucidation. The first enzyme to be crystallized and have its structure successfully solved was chicken egg lysozyme in 1965 [14, 15].
- Importantly, as well as the structure of the free enzyme, it was possible to crystallize lysozyme with a substrate analog bound in the active site. This structure allowed the proposal of a chemical mechanism for the enzyme, based on the positioning of groups around the site of substrate cleavage. The use of crystal structures with bound substrate and transition state analogs has helped to reveal the catalytic mechanisms of countless enzymes since.
- The structure of lysozyme was solved to a resolution of 2˚A; at this resolution, it is possible to accurately place the residue side chains and the plane of each peptide bond. However, individual atoms are not generally well resolved. ‘Atomic’ resolution (1.2˚A resolution or higher) allows the placement of atoms with fewer geometrical restraints and so gives a better picture of the ‘true’ protein structure.
- In recent years, advances in X-ray sources and cryocrystallography have led to increasing numbers of structures solved at these high resolutions Any structure, no matter what the resolution, contains a certain amount of error. Quite substantial errors, that have only been identified at a later date, have been found in some published protein structures.
- It has also been found that different areas of a crystal structure can have quite different amounts of error. Part of the reason for this lies in the physical nature of the crystal, some parts, particularly the loop regions, may be naturally flexible and so do not lie in a single conformation. This leads to these regions diffracting poorly and so the atoms within them being placed less accurately.
- Disorder in the crystal is measured by the temperature factor associated with each atom that specifies the positional variability of that atom. It has also been found that some important parts of protein structures, such as ligands bound within an enzyme, are prone to error because the geometrical restraints used in refining these regions are of a lower quality compared to those used to refine the protein itself.
Any two molecules have to collide for the reaction to occur along with the right orientation and a sufficient amount of energy. The energy between these molecules needs to overcome the barrier in the reaction. This energy is called as activation energy.
Enzymes are said to possess an active site. The active site is a part of the molecule that has the definite shape and the functional group for the binding of reactant molecules. The molecule binding to the enzyme is called as the substrate group. The substrate and the enzyme form an intermediate reaction with low activation energy without any catalysts.
\(reactant(1) + reactant(2) \rightarrow product\\ reactant(1) + enzyme \rightarrow intermediate\\ intermediate + reactant(2) \rightarrow product + enzyme\)
Factors Affecting Enzyme Activity
Generally, an increase in temperature increases the activity of enzymes. Because enzymes function in cells, the optimum conditions for most enzymes are moderate temperatures. At elevated temperatures, at a certain point activity decreases dramatically when enzymes are denatured. Purified enzymes in diluted solutions are denatured more rapidly than enzymes in crude extracts. Incubation of enzymes for long periods may also denature enzymes. It is more suitable to use short incubation time in order to measure the initial velocities of the enzyme reactions.
The International Union of Biochemistry recommends 30 °C as the standard assay temperature. Most enzymes are very sensitive to changes in pH. Only a few enzymes function optimally below pH 5 and above pH 9. The majority of enzymes have their pH-optimum close to neutrality. The change in pH will change the ionic state of amino acid residues in the active site and in the whole protein. The change in the ionic state may change substrate binding and catalysis. The choice of substrate concentration is also crucial because at low concentrations the rate is dependent on the concentration, but at high concentrations, the rate is independent of any further increase in substrate concentration.
Enzymatic catalysis relies on the action of amino acid side chains arrayed in the active center. Enzymes bind the substrate into a region of the active site in an intermediate conformation.
The active site is often a pocket or a cleft formed by the amino acids that participate in substrate binding and catalysis. The amino acids that make up the active site of an enzyme are not contiguous to one another along the primary amino acid sequence. The active site amino acids are brought to the cluster in the right conformation by the 3-dimensional folding of the primary amino acid sequence. Of the 20 different amino acids that make up protein, the polar amino acids, aspartate, glutamate, cysteine, Serine, histidine, and lysine have been shown most frequently to be active site amino acid residues. Usually, only two to three essential amino acid residues are directly involved in the bond leading to product formation. Aspartate, glutamate, and histidine are the amino acid residues that also serve as the proton donors or acceptors.
- Optimum T°
- Greatest number of molecular collisions
- human enzymes = 35°- 40°C
- body temp = 37°C
- Heat: increase beyond optimum T°
- Increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate H, ionic = weak bonds
- Denaturation = lose 3D shape (3° structure)
- Cold: decrease T°
- molecules move slower decrease collisions between enzyme & substrate
Changes in salinity: Adds or removes cations (+) & anions (–)
- Disrupts bonds, disrupts 3D shape
- Disrupts attractions between charged amino acids
- Affect 2° & 3° structure
- Denatures protein
- Enzymes intolerant of extreme salinity
- The Dead Sea is called dead for a reason
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