Enzyme Activity Control and Genetic Engineering Techniques
Enzyme Activity Regulation
Any substance that reduces the velocity of an enzyme-catalyzed reaction can be called an inhibitor. The inhibition of enzyme activity is one of the major regulatory mechanisms of the cell. It is of the following types:
Types of Enzyme Inhibition
Competitive Inhibition
Here, organic molecules compete with the substrate for binding at the active site of the enzyme. Such inhibitors show a close resemblance to the substrate of the enzyme. Instead of the enzyme-substrate complex, an enzyme-inhibitor complex is formed in such cases:
Enzyme + Inhibitor → Enzyme-Inhibitor Complex
An example of competitive inhibition is with succinic acid dehydrogenase (SDH). SDH oxidizes succinic acid to fumaric acid. The activity of succinic acid dehydrogenase declines rapidly when malonic acid is added. The distance between the two carboxyl groups is about the same in both compounds, and each fits the active site of SDH. As malonic acid has a structural resemblance to succinic acid, it acts as a competitive inhibitor. Competitive inhibition can be reversed by increasing the concentration of the substrate.
Competitive inhibitors are used to control bacterial pathogens. For instance, sulfa drugs act as competitive inhibitors of folic acid synthesis in bacteria as they substitute for p-aminobenzoic acid (PABA). These drugs are structurally related to PABA, a vital precursor in the microbial biosynthesis of folic acid, and inhibit the synthesis of the vitamin.
Non-Competitive Inhibition
Sometimes there is no competition for the active sites of an enzyme by an inhibitor; instead, it binds to some other site on the enzyme. As a result, the physical structure of the enzyme may be altered. This alteration prevents the formation of an enzyme-substrate complex, and the reaction does not occur. In non-competitive inhibition, an inhibitor binds to an enzyme regardless of whether or not the active site is occupied by the substrate. Many enzymes can be poisoned by non-competitive inhibition. For example, cyanide poisoning occurs because of the inhibition of cytochrome oxidase.
Uncompetitive Inhibition
Here, the inhibitor binds only to the enzyme-substrate complex and not to the free enzyme. This type of inhibition is rare in reactions that involve a single substrate but is more common in reactions with multiple substrates.
Feedback Inhibition
In many cases, the accumulation of the final product of a reaction causes the inhibition of the first step of the reaction. This is a useful mechanism because it prevents the excessive accumulation of products. For example, in Escherichia coli, the formation of the amino acid isoleucine is stopped when it has been formed in optimum quantity. The final product, isoleucine, acts as an inhibitor at the first stage of its synthesis.
Similarly, during glycolysis in respiration, hexokinase acts on glucose to form glucose 6-phosphate. Allosteric inhibition occurs by glucose 6-phosphate, thus decreasing the activity of the enzyme hexokinase.
Allosteric enzymes bear two types of sites: active and allosteric. The end product combines with the allosteric site of the enzyme and checks its catalytic activity. The product that acts as the inhibitor brings about conformational changes in the active sites of enzymes, resulting in the reduction of catalytic activity. Jacob and Monod termed this phenomenon allosteric transition.
Genetic Engineering and Vectors
Genetic engineering is a technique by which new combinations of genetic material can be made in the laboratory using DNA from two or more organisms. The various types of vectors used in genetic engineering are as follows:
Types of Vectors in Genetic Engineering
Plasmids as Vectors
Plasmids are the most widely used vectors for genetic engineering in bacteria. They are circular, double-stranded DNA molecules that occur as extrachromosomal DNAs and are self-replicating. A cell may contain 10-20 plasmids. Plasmids possess a replication control system that maintains them in the bacterium at a characteristic level.
There are two general types of plasmids: single-copy plasmids (maintained as a single copy per cell) and multicopy plasmids (existing in about 1000 copies per cell). These plasmids are often used to provide cloning vectors.
The plasmid vector is isolated from the bacterial cell and cleaved at one site by a restriction endonuclease. This creates a linear DNA stretch. The two ends of this linear DNA are joined with the help of DNA ligase to the ends of the foreign DNA to be inserted. This re-creates a circular DNA, forming a chimeric or recombinant DNA. This recombinant DNA is then transferred to a bacterium, where it replicates and perpetuates indefinitely.
Many vectors have been found that can exist in both eukaryotic cells and Escherichia coli. Such vectors, known as shuttle vectors, bear two types of origin of replication and have selectable marker genes, one acting in eukaryotic cells and the other in E. coli. An example is the shuttle vector of yeast episomal plasmid (YEp).
In plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens, called the Ti plasmid, has been found suitable to act as a vector.
An ideal cloning plasmid vector should have a low molecular weight, the ability to readily confer with a selectable marker, and a large number of restriction enzyme sites.
Phages as Vectors
Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. Two phages extensively used for this purpose are lambda (λ) and M13.
Lambda phage DNA is a linear, double-stranded molecule. Wild-type phage DNA contains several target sites for most commonly used restriction enzymes, making it unsuitable as a direct vector. Therefore, derivatives of the wild-type phage have been developed. These derivatives either have a single target site in their DNA where foreign DNA can be inserted (called insertional phage vectors) or have a pair of sites defining a DNA fragment that can be removed and replaced by foreign DNA (called replacement phage vectors).
Many vector derivatives of both insertional and replacement types have been produced recently for use, and most have been developed for use with EcoRI, BamHI, or HindIII restriction enzymes. Their application can be extended to other restriction enzymes by the use of linker molecules.
The phage head can accommodate only about 5% more than its normal complement of DNA, which prevents excessively long foreign DNA from being packaged into it. To overcome this problem, a fragment of phage DNA that does not carry essential phage genes is removed to increase the space within the phage DNA, and this space is then replaced by foreign DNA.
The chimeric or recombinant DNA is packaged into the phage ‘head’ coat in vitro. The purpose of in vitro packaging is to supply the ligated recombinant DNA with high concentrations of phage head precursors, packaging proteins, and phage tails.