PubMed · 775084
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P A Ward. 1976. Leukotactic responses.. https://pubmed.ncbi.nlm.nih.gov/775084/
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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.
Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.
The effects of a range of metallopeptidase inhibitors on the activities of the porcine kidney cell surface zinc aminopeptidases, aminopeptidase A (AP-A; EC 3.4.11.2), aminopeptidase N (AP-N; EC 3.4.11.7) and aminopeptidase W (AP-W; EC 3.4.11.16), have been directly compared. Amastatin and probestin were effective against all three aminopeptidases, with the concentration of inhibitor required to cause 50% inhibition (I50) in the low micromolar range (I50 = 1.5-20 microM), except for probestin with AP-N which displayed an I50 of 50 nM. Actinonin failed to inhibit significantly either AP-A or AP-W, and thus can be considered a relatively selective inhibitor (I50 = 2.0 microM) of AP-N. In contrast, bestatin was a relatively poor inhibitor of AP-N (I50 = 89 microM) and failed to inhibit AP-A, but was more potent towards AP-W (I50 = 7.9 microM). Thus, some of the observed chemotherapeutic actions of bestatin may be due to inhibition of cell-surface AP-W. A number of other metallopeptidase inhibitors, including inhibitors of endopeptidase-24.11 (EC 3.4.24.11) and membrane dipeptidase (EC 3.4.13.11), and the carboxylalkyl and phosphoryl inhibitors of angiotensin converting enzyme (EC 3.4.15.1) failed to inhibit significantly AP-A, AP-N or AP-W. However, AP-W was inhibited with I50 values in the micromolar range by the sulphydryl converting enzyme inhibitors rentiapril (I50 = 1.6 microM), zofenoprilat (I50 = 7.0 microM) and YS 980 (I50 = 17.7 microM). Neither AP-A nor AP-N were affected by these sulphydryl compounds. Inhibition of AP-W may account for some of the side effects noted with the clinical use of the sulphydryl converting enzyme inhibitors. The availability of compounds which are totally selective for AP-W over any of the other mammalian cell surface zinc aminopeptidases may aid in identifying endogenous substrates, and thus physiological or pathophysiological role(s) of AP-W.