You Gotta Know These Enzymes
- DNA polymerases catalyze the synthesis of DNA from a DNA template, particularly during DNA replication (in which one DNA molecule makes two identical copies, typically during the S phase of the cell cycle). The polymerase reacts a growing DNA strand with a nucleoside triphosphate (ATP, GTP, CTP, and TTP), resulting in a DNA strand one base longer and an ion of pyrophosphate (P2 O74−). There are several enzymes in the DNA polymerase family: DNA Polymerase I (or Pol I) participates in DNA replication in prokaryotes; it was discovered by Arthur Kornberg in E. coli. In eukaryotic cells, DNA polymerases α, δ, and ε (alpha, delta, and epsilon) are the polymerases most involved in nuclear DNA replication. DNA polymerases synthesize new DNA in the 5′ to 3′ direction, but they also possess a 3′ to 5′ exonuclease activity to fix mistakes made during replication (a process called proofreading). DNA polymerases can quickly add nucleotides to a DNA strand because of their ability to slide along a template without slipping off, a characteristic known as processivity. Taq polymerase, a specific DNA polymerase from the bacteria Thermus aquaticus, is stable at high temperatures and is thus used in polymerase chain reactions (PCRs) in molecular biology laboratories.
- RNA polymerase catalyzes the formation of single-stranded RNA from a DNA template. In eukaryotes, RNA polymerase II is the member of the RNA polymerase family that synthesizes precursors of mRNA during transcription. In prokaryotes, RNA polymerase is bound to a sigma factor, which binds to the core promoter region in which transcription is initiated. In eukaryotes, the function of the sigma factor is performed by a group of proteins called general transcription factors, which associate with RNA polymerase to form the transcription preinitiation complex. The RNA polymerase complex (known as a holoenzyme) then switches from a closed complex to an open one, unwinding a portion of DNA to form the transcription bubble. Early on during RNA synthesis, RNA polymerase must escape the promoter; if it fails to do so, short RNA transcripts are generated in a process called abortive initiation.
- Reverse transcriptase catalyzes the formation of complementary DNA (cDNA) from an RNA template. It is used by retroviruses such as HIV in order to replicate their genomes; a class of anti-HIV drugs known as reverse transcriptase inhibitors (including AZT) interfere with the function of the enzyme. Reverse transcriptase is also used by retrotransposons, which are genomic DNA sequences that copy and paste themselves into different locations using an RNA intermediate. In the laboratory, reverse transcriptase is used in RT-PCR, a variant of the polymerase chain reaction that aims to measure the expression levels of different genes.
- Telomerase adds a repeat sequence called a telomere to the end of chromosomes; telomeres shorten during each round of cell division, a phenomenon that is correlated with cellular aging (senescence). The telomerase complex contains a subunit called TERT, a reverse transcriptase enzyme that carries its own RNA molecule, which it uses as a template to add the six-nucleotide repeating sequence TTAGGG to the ends of chromosomes. Telomerase activity is regulated by shelterin, a protein complex that protects telomeres.
- Rubisco (sometimes capitalized RuBisCO, short for Ribulose-1,5-bisphosphate carboxylase/oxygenase) is an enzyme involved in the light-independent/dark reactions of photosynthesis, part of a pathway known as the Calvin cycle. The enzyme catalyzes the carboxylation (addition of C O2) of RuBP to form two molecules of glycerate-3-phosphate (G3P, or 3-PGA). However, oxygen (O2) can compete with C O2 for binding to Rubisco, resulting in an energy-wasteful side reaction called photorespiration. By some estimates, Rubisco is the most abundant enzyme on Earth.
- Catalase decomposes hydrogen peroxide into water and oxygen. It is found in virtually every organism that is exposed to oxygen, and is crucial for preventing cells from experiencing damage from reactive oxygen species (such as hydrogen peroxide, superoxide, the hydroxyl radical, and singlet oxygen, which can all damage DNA and participate in unwanted reactions with various proteins). In eukaryotic cells, catalase is localized to an organelle called the peroxisome. Catalase has an incredibly high turnover number (the number of chemical reactions that an enzyme can catalyze in one second): it can decompose millions of hydrogen peroxide molecules per second. In fact, it is so fast that it is termed catalytically perfect: its rate of reaction is so fast that the slowest process limiting its speed is the diffusion of molecules in and out of the active site. Microbiologists can identify bacteria that express catalase (which is most bacteria, with Streptococcus and Enterococcus as notable exceptions) via the catalase test: a drop of hydrogen peroxide is added to a microscope slide, and then a colony of bacteria is touched to it. If it bubbles, then the bacteria is catalase-positive.
- ATP synthase catalyzes the formation of a molecule of ATP from adenosine diphosphate (ADP) and inorganic phosphate (P O43−). ATP synthase is a molecular machine with two subunits: FO (‘O’ for “oligomycin,” which inhibits the enzyme) and F1, the latter of which rotates like a motor while producing ATP. ATP synthase is “powered” by a gradient of protons (H+) across the inner mitochondrial membrane or thylakoid membrane. This proton gradient, sometimes called the proton motive force, is generated by the action of the electron transport chain. Paul Boyer elucidated the mechanism of ATP synthase and John Walker crystallized the F1 domain (at the time the largest asymmetric protein structure known); the two shared the 1997 Nobel Prize in Chemistry for their work.
- Amylase catalyzes the hydrolysis of starch into simple sugars during digestion. It is present in the saliva of humans and other mammals, as well as in the pancreas (both of these forms of amylase are alpha-amylases). Amylase specifically cleaves the α-1,4-glycosidic bonds between glucose monomers in starch. Amylase was first isolated in 1833 by French chemists who called it “diastase,” establishing the use of “-ase” as a suffix in the names of enzymes.
- Pepsin breaks down proteins into peptides and amino acids during digestion. Its precursor (proenzyme or zymogen) pepsinogen is produced by gastric chief cells in the stomach and is most active in the acidic environment of gastric acid (pH between 1.5 and 2.5). Like the other digestive enzymes chymotrypsin and trypsin, pepsin is an endopeptidase, meaning that it cuts internal peptide bonds within the protein.
- Cas9 is an enzyme that is used in the CRISPR technique for gene editing. Native to Streptococcus pyogenes, Cas9 checks the spacer region of a guide RNA (gRNA) for any sites complementary to a piece of foreign DNA; if there are any such complementary sites, Cas9 cuts the foreign DNA. Cas9 thus functions like an “immune system” for bacteria, protecting them against bacteriophage DNA and DNA from foreign plasmids. By engineering guide RNAs with specific sequences, Cas9 can be used to cut DNA in specific locations, allowing for gene editing. Two residues in the RuvC and HNH domains of Cas9 can be mutated to alanine to generate a “dead” variant of Cas9 (dCas9), which has no cutting (endonuclease) activity; this variant has been used to study how Cas9 binds to DNA.
- Restriction enzymes (or restriction endonucleases) cut DNA into fragments at specific recognition sites, most of which are palindromic. The product of a restriction digest is either two strands of even length (a blunt end), or one strand that is slightly longer than the other (a sticky end). An example of a restriction enzyme that produces a blunt end is SmaI, whereas sticky ends are produced by the restriction enzyme EcoRI. Many binding sites for restriction enzymes are found on plasmids, which are often used during molecular cloning.
This article was contributed by ÎÞÓǶÌÊÓƵ editor Auroni Gupta.