3.1.1. Enzyme in Biomolecular Ladder
Biomolecular Ladder. Most of the chemical components of living organisms are organic compounds of carbon, many also containing oxygen and nitrogen. Although each living species contains various combination of these biomolecules, the diversity can be reduced to a few building blocks of common structure. It is possible to organize these building blocks on a hierarchical ladder, according to molecular weight (Fig. 3.1). The bottom of' the ladder is occupied by the low-molecular-weight gases (oxygen, nitrogen and carbon dioxide) and by water. These molecules together with the monoatomic ions, in particular Na+, K+, Mg2+, Ca2+, Cl- and the elements P, S, Mn, Fe, Co, Cu and Zn are most common in further involvement as progress is made up each rung of the molecular-weight ladder. The monitoring of these ions is frequently used to follow the metabolic state of a patient under care and is commonly achieved using ion selective electrode sensors, where the recognition surface of the sensor is provided by a membrane, containing a molecule with a selectivity for the target ion (see Chapter 2) . The oxygen and carbon dioxide electrodes f'orm two of the well-established base sensors to which analyte specific reactions have been linked via a biorecognition macromolecule. Note where 'protein', 'antibody', and 'enzyme complex' lie in the ladder. These are the major molecules with molecular-recognition capabilities that are utilized in biosensor development.
Metabolism Point of View. The food that we eat consists mainly of proteins, carboydrates (or polysaccharides), and lipids. These nutrients are broken down in our body with the aid of oxygen that we breathe. This breakdown process called 'catabolism' produces the basic building blocks of the body - amino acids, simple sugars, and fatty acids. Also, energy is produced (and stored in the form of ATP) during the catabolism which is essentially a partial oxidation process. These building blocks and energy are then used to make the main components of our body - proteins, polysaccharides, and lipids. These components are used to make supramolecular assemblies such as enzyme complexes, ribosomes (this is where proteins are made), etc. The synthesis processes are collectively called the 'anabolism' which is dictated by the genetic codes written on the genes of the cell (the DNA helix).
Protein Structure. Proteins are polymers of amino acids. All amino acids have a common structure - a carbon molecule with a carboxyl group (-COOH), an amino group (-NH2), and R group (Fig. 3.2a). It is the R group which
Fig. 3.1. Biomolecular ladder.
(a) Basic structure of amino acid
(b) Formation of peptide bond
(c) Limited rotation in peptide chain
(d) non-covalent inter- and intra-molecular bonds in peptide strands.
Fig. 3.2. (a) Basic structure of amino acid; (b) formation of peptide bond; (c) limited rotation in the peptide strand; (d) non-covalent inter- and intra-molecular bonds in peptide strands.
makes each amino acid unique. For example when R = CH3, it is called alanine. The amino acids are connected together by a peptide bond, which is the bond between -COOH of one amino acid and -NH2 of the other (Fig.3.2b). The proteins are made in the riobosomes (an organel made of ribosomal RNAs) of the cells. The strucutre and the function of the proteins depend not only on the amino acicd sequence (called the primary structure), but also on the conformation (the secondary, tertiary, and quaternary structures). The peptide bond and the disulphide bridges impart certain restrictions on the structure (Fig. 3.2c). The peptide strands are further organized by interactions between residue side chains. The nature of the bonds include hydrogen bond, ionic bond, and hydrophobic bond (Fig. 3.2d). An example of an enzyme is shown in Fig. 3.3.
Ionic Property of Proteins. The net result of all the interactions between the amino acids is that there is a spontaneous folding of a protein to give a unique structure. All the amillo acids have at least two groups capable of existing in ionic form. The -(carboxyl groul), -COOH, can lose H+ to become COO- . The reaction is pH dependent, and is characterized by a pKa typically in the range 2 to 3. Similarly the -amino group, NH2, can be protonated to give NH3+ and has a pKa value of about 10. Therefore, between about pH 4 and 9 the amino acid exists as a dipolar ion zwitterion with little net charge (Fig. 3.3d). At the isoelectric point, pI, the protein has no net charge, and it will not move in an electric field.
Ioselectrical Point. Where R contains no ionizable groups,
The movement of the amino acids under the influence of an electric field allows their separation and identification; it can be used as a powerful assay technique. The dual polarity feature accounts for many of the properties of amino acids, e.g. the large dipole moments, the high solubility in water and low solubility in organic solvents
Ionic Behavior of Proteins. By analogy, it would be expected that each peptide strand would also contain at least two ionizable groups, but since the -carboxyl or -amino groups are now involved in peptide-bond formation, they are not available for ionization. The zwitterionic behaviour is therefore more restricted to the terminal amino group and the terminal carboxyl group. These groups are considerably further away from one another than they would be in free amino acids and so the electrostatic interactions between them are diminished and their pKa values are lower than in the -amino acid. It follows that the groups in proteins
that are principally involved in acid-base equilibria are the side-chain
R groups. Ordinarily there may be 50-60 titratable groups per 100,000 molecular
weight of protein. Therefore, the titration curves for proteins are complex
and difticult to interpret.
Molecular Recognition by Enzyme. The biorecognition properties of protein molecules will depend almost entirely on the amino acids of the exposed surfaces. Weak non-covalent interactions can occur between the residues on the exposed surfaces of the protein and other non-protein molecules (Fig. 3.4). If a sufficient number of these weak bonds are formed simultaneously with the incoming molecule, then the molecule can bind tightly to the protein. Obviously for this to occur the molecule must fit precisely into the binding site on the protein surface. This feature is analogous to the recognition surface of the model biosensor.
Enzyme Complexes. One of the most important functions of proteins is to act as catalysts or enzymes for chemical reactions. These enzymes are able to stabilize the transition state between a substrate and its products by interactions at the binding site (of the substrate). The activation of most biochemical reactions fall in the range of 40-80 kcal/mol without the enzyme. The enzymes lower this activation energy. For example, the splitting of H2O2 takes 75.4 kJ/mol without the enzyme, whereas a catalase enzyme lowers the activation energy to 23 kJ/mol. Substrate specificity by the enzyme is provided by the surface interactions and this characterisitcs is exploited in the development of enzylne-based biosellsors. The none-covalent binding of the enzyme substrate transition state lowers the activation energy for the reaction and thus catalyzes the reaction.