Biochemistry: Enzyme Inhibition and Antibiotics

Regulation of Enzyme Activity

• Enzyme regulation is crucial as cellular processes continually produce large amounts of an enzyme and products if not regulated
• General mechanisms involved in regulation include proteolytic enzymes and zymogens, covalent modification of enzymes, and regulation of enzyme activity by various substances produced within a cell
• Allosteric enzymes have quaternary structure, with at least two binding sites: substrate and regulator binding sites, which are distinct from each other

Antibiotics that Inhibit Enzyme Activity

• Antibiotics inhibit specific enzymes essential to life processes of bacteria
• Two families of antibiotics considered are sulfa drugs and penicillin's
• Sulfa drugs exhibit antibiotic activity by competitively inhibiting enzymes responsible for converting PABA to folic acid in bacteria
• Folic acid deficiency retards bacterial growth and eventually kills them
• Sulfa drugs don't affect humans because we absorb folic acid from our diet

Penicillin's

• Penicillin's were accidentally discovered by Alexander Fleming in 1928
• Several naturally occurring penicillin and numerous synthetic derivatives have been produced
• All have structures containing a four-membered Beta-lactam ring fused with a five-membered thiazolidine ring
• Penicillin's selectively inhibit transpeptidase by covalent modification of a serine residue
• Transpeptidase catalyzes the formation of peptide cross-links between polysaccharide strands in bacterial cell walls

Vitamins

• Vitamins are components of coenzymes
• Fat-soluble vitamins: A, D, E, and K
• Water-soluble vitamins: B vitamins, comprising thiamin (vitamin B1), Riboflavin (vitamin B2), Niacin (vitamin B3), Vitamin B6, Folate (folic acid), Vitamin B12 (cobalamin), Pantothenic acid (vitamin B5), and Biotin
• B Vitamins exhibit structural diversity

Nucleic Acids

• RNA (ribonucleic acid) is found mainly in the cytoplasm of living cells
• DNA (deoxyribonucleic acid) is found mainly in the nucleus of living cells
• DNA and RNA are polymers consisting of repeating subunits called nucleotides, which are made of three components: heterocyclic base, sugar, and phosphate
• Heterocyclic bases: pyrimidine and purine
• Pyrimidine bases: uracil (U), thymine (T), and cytosine (C)
• Hydrogen bonds: U hydrogen bonds to A, and G hydrogen bonds to C, forming a set of complementary base pairs

DNA Structure

• DNA is one of the largest molecules known, containing between 1 and 100 million nucleotide units
• Nucleotides in DNA are linked by phosphate groups that connect the 5' carbon of one nucleotide to the 3' carbon of the next
• The nucleic acid backbone is a sequence of sugar-phosphate groups, which differ only in the sequence of bases
• The base sequence of a DNA strand is always written from the 5' end to the 3' end

DNA Replication

• The process of DNA replication begins with the unwinding of the nucleic acid strands at a specific point called the replication fork
• An RNA primer attaches to the DNA at the point where replication begins
• The Okazaki fragments are synthesized by DNA polymerase along the lagging strand as the replication fork moves
• The Okazaki fragments are joined by DNA ligase, resulting in two new DNA molecules

Polymerase Chain Reaction (PCR)

• PCR is an important laboratory technique that mimics the natural process of replication
• A small quantity of target DNA, a buffered solution of DNA polymerase, the cofactor MgCl2, the four nucleotide building blocks, and primers are added to a test tube
• The mixture goes through several three-step replication cycles: heat (94-96°C) is used for one to several minutes to unravel DNA into single strands; the primers bind to the DNA strands and serve as starting points for new chain growth; the mixture is cooled, allowing the primers to anneal to the DNA strands

The Formation of Recombinant DNA

• Restriction enzymes, found in a wide variety of bacterial cells, catalyze the cleaving of DNA molecules, except for a few specific types
• Restriction enzymes act at sites on DNA called palindromes, where two strands have the same sequence but run in opposite directions
• Restriction enzymes are used to break DNA up into fragments of known size and nucleotide sequence, which can then be spliced together with DNA ligases