PubMed · 6141621
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D Tsuru. 1983. [Pyroglutamyl peptidase].. https://pubmed.ncbi.nlm.nih.gov/6141621/
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INTRODUCTION: ER aminopeptidase 1 (ERAP1) is a polymorphic intracellular aminopeptidase with key roles in antigen presentation and adaptive immune responses. ERAP1 allotype 10 is highly protective toward developing some forms of autoimmunity and displays unusual functional properties, including very low activity versus some substrates. METHODS: To understand the molecular mechanisms that underlie the biology of allotype 10, we studied its enzymatic and biophysical properties focusing on its unique polymorphisms V349M and Q725R. RESULTS: Compared to ancestral allotype 1, allotype 10 is much less effective in trimming small substrates but presents allosteric kinetics that ameliorate activity differences at high substrate concentrations. Furthermore, it is inhibited by a transition-state analogue via a non-competitive mechanism and is much less responsive to an allosteric small-molecule modulator. It also presents opposite enthalpy, entropy, and heat capacity of activation compared to allotype 1, and its catalytic rate is highly dependent on viscosity. Polymorphisms V349M and Q725R significantly contribute to the lower enzymatic activity of allotype 10 for small substrates, especially at high substrate concentrations, influence the cooperation between the regulatory and active sites, and regulate viscosity dependence, likely by limiting product release. CONCLUSIONS: Overall, our results suggest that allotype 10 is not just an inactive variant of ERAP1 but rather carries distinct enzymatic properties that largely stem from changes at positions 349 and 725. These changes affect kinetic and thermodynamic parameters that likely control rate-limiting steps in the catalytic cycle, resulting in an enzyme optimized for sparing small substrates and contributing to the homeostasis of antigenic epitopes in the ER.
Proteolytic activities were investigated in sorghum-based togwa prepared by natural fermentation and using starter cultures previously isolated from the native product, i.e., Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum, Pediococcus pentosaceus, and Issatchenkia orientalis in coculture with either L. brevis or L. plantarum. Both proteinase and aminopeptidase activities were substantially higher in naturally fermented togwa than in those with starters (14-30%, 12-70%, respectively). A variable but substantial part of the proteinase activity followed the particulate fraction of togwa; aminopeptidase activity was mainly in that fraction. The breakdown of relatively high molecular mass protein (64 kDa) in togwa was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); the products were mainly in the 14-30 kDa range. Reversed-phase fast-protein liquid chromatography (RP-FPLC)-protein/peptide patterns changed during fermentation with some variation between togwa of different cultures. Supplementation of gruel with malt increased the concentration of total protein [from 9.5% to 11.0% (w/w) on dry weight basis)] and of most of the free amino acids. Fermentation had no effect on total protein content; however, the concentration of most of the amino acids was reduced, except for the proline content that increased. Natural fermentation also increased the concentration of glutamic acid and ornithine. Fermentation by P. pentosaceus increased aspartic acid, while L. cellobiosus, L. fermentum, and L. brevis in coculture with I. orientalis increased the concentration of citrulline.
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.