Ligase, definition

Since all ligase reactions are coupled by definition, the reactions that are coupled are shown. 

Because of coupling (see Chapter 7) there are relationships between the thermodynamic properties of reactions in some of the EC classes. All oxidoreductase reactions can be considered to be coupled reactions because each one can be divided into two, or in a some cases, three half reactions that do not share atoms but are connected by formal electrons. Transferase reactions can each be considered to result from the coupling of two oxidoreductase reactions or two hydrolase reactions. Fifteen examples are discussed in reference (6). Each of the coupled reactions contributes its A, G ° and A, A h to the coupled reaction. Hydrolase reactions and isomerase reactions are never coupled reactions. Some lyase reactions are coupled. Ligase reactions are all coupled by definition because they join together two reactions with the hydrolysis of a pyrophosphate bond in ATP or a similar triphosphate. A spectacular example of coupling is provided by EC 6.3.5.4 because there are seven reactants. This never happens in chemistry. 

T4 DNA ligase (10 USB units/pl, note that the unit definition varies among different suppliers)... 

The LCR conditions shown serve as one, specific example. For individual applications, conditions must be optimized. We have found the critical optimization factors to be probe concentration, annealing temperature, cycle number, and ligase concentration. Any commercially available thermophilic DNA ligase should be suitable with a compatible buffer. We have had good success with ligase purified from Tkermus thermophilis. Since there is no universally accepted enzyme unit definition, the optimal amount of enzyme to use must be determined experimentally. 

The aminoacyl-tRNA ligases, of course, provide the mechanism by which an amino add is activated in the presence of ATP and an activating em me specie for that amino add. The reaction is a reversible one, and can be diagramm as follows [reaction (I)], bearing in mind that our former colleague Robert Loftfield may be correct in regarding the path from free amino acid to aminoacyl-tRNA in some instances to represent a concerted reaction mechanism, without a definitive intermediate. 

DNA ligase activities can be assayed by various methods involving either partial or whole ligation reactions. The principal assay methods and unit definitions are as follows. 

Unit definition One ligase unit is that amount of enzyme which catalyzes 50% ligation of 1 /tg of A DNA HmdIII fragments in 30 min at 16 C. One cohesive-end ligation unit is equivalent to 0.015 Weiss units or 0.0025 Modrich-Lehman units. 

Unit definition One ligase unit is the amount of enzyme that converts 1 nmol of [5 - P]rA2o to a phosphatase-resistant form in 30 min. 

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