The active sites in PepA and in bovine lens LAP have been found to be similar. Shown in the picture below is the proposed model for the active site of LAP-A in tomato based on the work of Strater et al. It is also known that the biochemistry of the LAPs from these three kingdoms is very similar. PepA, bovine lens LAP, and LAP-A preferentially cleave N-terminal leucine, arginine, and methionine residues. These enzymes are all metallopeptidases requiring divalent metalcations for their enzymatic activity Enzymes are active in the presence of Mn+2, Mg+2 and Zn+2. These enzymes are also known to have high pH and temperature optima. At pH 8, the highest enzymatic activity is seen at 60 °C. PepA, bovine lens LAP and LAP-A are also known to form hexamers in vivo. The Gu et al. from 1999 demonstrated that six 55kDA enzymatically inactive LAP-A protomers come together to form the 353kDa bioactive LAP-A hexamer. Structures of the bovine lens LAP protomer and the biologically active hexamer have been constructed can be found through Protein Data Bank.
Mechanism(s)
Historically, the mechanisms of carboxypeptidases and endoprotease have been much more well-studied and understood by researchers. Work within the past two decades has provided vital knowledge regarding the mechanisms of aminopeptidases. The mechanism of bovine lens LAP and PepA have been elucidated, however, the exact mechanism of tomato LAP-A is unknown at this time. A search of current literature does not indicate that new research is underway to determine the exact mechanism of LAP-A. Based on the biochemical similarities of the LAPs between kingdoms, the mechanism of LAP-A may be similar to bovine lens LAP and PepA.
In order to survive, plants must be able to respond to many biotic and abiotic stresses, including pathogen attack, piercing/sucking insects, herbivory, and mechanical wounding. These stresses activate specialized signal transduction pathways, which are specific to the stressor and the amount of tissue damage inflicted. Similar to mechanical wounding, chewing insects, such as the tobacco hornworm, cause extensive tissue damage activating the jasmonic acid -mediated response. This JA-mediated response revolves around the octadecanoid pathway, which is responsible for the synthesis of JA and several other potent signaling molecules, and ends in the regulation of two sets of genes whose expression changes over time. The early genes amplify the wounding signal and can be detected 30 minutes to 2 hours after damage. Late gene expression can be seen 4–24 hours after wounding. Products of late-response genes act as deterrents to chewing-insect feeding, often by decreasing the nutritional value of the food ingested or interfering with insect gut function. For example, serine proteinase inhibitors interfere with digestive proteases in the insect gut and polyphenol oxidases act to decrease the nutritive value of plant leaves after ingestion by herbivores. Please see the Picture 3 for a summary of the wound response in tomato. The plant response in this octadecanoid pathway is similar to mammalianprostaglandin and leukotriene pathways. This particular pathway is inhibited by salicylic acid.
Octadecanoid pathway
, a product of the octadecanoid pathway in some solanaceous plants, has been shown by Fowler et al. to have a regulatory role in the late wound response of tomato. Experiments were conducted using three genotypes of tomato plants: wildtype, plants that were silenced for LAP-A, and LapA-OX that constitutively expressed LAP-A. Late-gene expression was inhibited in wounded LapA-SI plants, and the LapA-SI plants were also more susceptible to tobacco hornworm feeding, relative to wildtype plants. In comparison, the wounded LapA-OX leaves exhibited heightened levels of late gene RNA accumulation, an increased resistance to herbivory, and extended expression of late wound-response genes. These data suggest that LAP-A functions in regulating both the intensity and the persistence of the late wound response. However, unwounded LapA-OX did not accumulate late gene RNA transcripts, suggesting that presence of LAP-A alone is not sufficient to induce late gene expression. LAP-A is the first plant aminopeptidase shown to have a regulatory role in signal transduction pathway.
Osmoregulation
LAP proteins are expressed in a variety of marine organisms as a method of coping with the osmotic threat high salinity poses to the cell. During bouts of high salinity, LAP begins the catalysis of proteins in order to release amino acids into the cell in an attempt to balance the high ion concentrations in the external environment.
