<P> Diisopropyl fluorophosphate (DIFP) is an irreversible inhibitor that blocks the action of serine protease . When it binds to the enzyme a nucleophilic substitution reaction occurs and releases one hydrogen fluoride molecule . The OH group in active site acts as a nucleophile to attack the phosphorus in DIFP and form a tetrahedral intermediate and release a proton . Then the P-F bond is broken, one electron is transferred to the F atom and it leaves the intermediate as F anion . It combines with a proton in solution to form one HF molecule . Now there is a covalent bond formed between the active site and DIFP, so the serine side chain is no longer available to the substrate . </P> <P> Identification of active sites is crucial in the process of drug discovery . The 3 - D structure of the enzyme is analysed to identify active sites and design drugs which can fit into them . Proteolytic enzymes are targets for some drugs, such as protease inhibitors, which include drugs against AIDS and hypertension . These protease inhibitors bind to an enzyme's active site and block interaction with natural substrates . An important factor in drug design is the strength of binding between the active site and an enzyme inhibitor . If the enzyme found in bacteria is significantly different from the human enzyme then an inhibitor can be designed against that particular bacterium without harming the human enzyme . If one kind of enzyme is only present in one kind of organism, its inhibitor can be used to specifically wipe them out . </P> <P> Active sites can be mapped to aid the design of new drugs such as enzyme inhibitors . This involves the description of the size of an active site and the number and properties of sub-sites, such as details of the binding interaction . Modern database technology called CPASS (Comparison of Protein Active Site Structures) however allows the comparison of active sites in more detail and the finding of structural similarity using software . </P> <Table> <Tr> <Th> </Th> <Th> Example </Th> <Th> Mechanism of action </Th> </Tr> <Tr> <Td> Anti-bacterial agent </Td> <Td> Penicillin </Td> <Td> Bacteria cell wall is composed of peptidoglycan . During bacterial growth the present crosslinking of peptidoglycan fibre is broken, so new cell wall monomer can be integrated into the cell wall . Penicillin works by inhibiting the transpeptidase which is essential for the formation of crosslinks . So the cell wall is weakened and will burst open due to turgor pressure . </Td> </Tr> <Tr> <Td> Anti-fungi agent </Td> <Td> Azole </Td> <Td> Ergosterol is a sterol that forms the cell surface membrane of the fungi . Azole can inhibit its biosynthesis by inhibiting the Lanosterol 14 alpha - demethylase . So no new ergosterol is produced and harmful 14α - lanosterol is accumulated within the cell . Also, azole may generate reactive oxygen species . </Td> </Tr> <Tr> <Td> Anti-viral agent </Td> <Td> Saquinavir </Td> <Td> HIV protease is needed to cleave Gag - Pol polyprotein into 3 individual proteins . So they can function properly and start viral packaging process . HIV protease inhibitor like Saquinavir inhibits it so no new mature viral particle can be made . </Td> </Tr> <Tr> <Td> Insecticides </Td> <Td> Physostigmine </Td> <Td> In animal nervous system, Acetylcholinesterase is required to breakdown neurotransmitter acetylcholine into acetate and choline . Physostigmine binds to its active site and inhibits it, so impulse signal cannot be transmitted through nerves . Insects die as they lose control of muscle and heart, </Td> </Tr> <Tr> <Td> Herbicides </Td> <Td> Cyclohexanedione </Td> <Td> Cyclohexanedione targets the Acetyl - CoA carboxylase which is involved in the first step of the fat synthesis: ATP - dependent carboxylation of acetyl - CoA to malonyl - CoA. Lipid is important in making up the cell membrane . </Td> </Tr> </Table>

Which of the following types of regulation involves binding to the active site