PubMed · 69246
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H Umezawa. Recent studies on bleomycin.. https://pubmed.ncbi.nlm.nih.gov/69246/
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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.
8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG. C-containing duplex oligo with a very low apparent k(cat) of 0.1 min(-1) at 37 degrees C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis-Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased approximately 5-fold and Michaelis-Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP.C pair (K:(d) approximately 2.8 nM) was substantially higher than for its substrate 8-oxoG.C pair (K:(d) approximately 23. 4 nM) and the affinity for its final ss-elimination product was much lower (K:(d) approximately 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.
Vacuolar aminopeptidase 1 is transported to the vacuole by cytoplasmic double-membrane vesicles, the nonclassic Cvt pathway. The cytosolic protein dodecamerizes and is enclosed in a double-membrane vesicle, which is transported to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of aminopeptidase 1 is removed. This pathway resembles autophagy, and most proteins identified to function in the Cvt pathway are also required for autophagy and vice versa. The cytosolic precursor protein and the matured vacuolar protein form a homododecameric complex, and only this complex has enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of vacuolar aminopeptidase 1 based on its enzymatic activity. The sensitivity of this assay made it possible for us to search for mutants under conditions where autophagy is down-regulated, and we describe two new mutants defective in the biogenesis pathway of vacuolar aminopeptidase 1. Mutants are defective in dodecamerization of pApe1p and in Cvt vesicle formation. Complex assembly and transport vesicle formation appear to be linked processes. This mechanism can control the potentially harmful cytoplasmic proteolytic activity and could be the driving force for this nonclassic mechanism of vacuolar enzyme transport.