Abstract
An apparently unique enzyme, designated protein NH2-terminal asparagine deamidase (PNAD), that specifically converts NH2-terminal asparagine residues of peptide and protein substrates to aspartic acid, has been isolated to homogeneity from porcine liver by an eight-step procedure. PNAD is a relatively low abundance protein, is readily solubilized, and exists as a monomeric species of approximately 33 kDa. PNAD does not act on internal asparagine residues and requires a free N alpha-amino group. It has reduced or no activity on NH2-terminal asparagine dipeptides and no activity toward free asparagine or asparagine amide. It does not act on any NH2-terminal glutamine substrates. PNAD does not show a strong pH dependence suggesting that the enzyme can act equally well on substrates with ionized or unionized alpha-amino groups. The properties and specificity of PNAD are consistent with those expected for the enzyme required for the ubiquitin-dependent turnover of intracellular proteins that initiate with Met-Asn-. Such proteins should be N alpha-acetylated on the retained initiator methionine and can subsequently be modified by the removal of N-acetyl methionine by acylaminoacid hydrolase. Conversion of the resulting NH2-terminal asparagine to aspartic acid by PNAD would render the protein susceptible to arginylation, polyubiquitinylation and degradation as specified by the N-end rule.
Highlights
NH,terminal asparagine deamidase (PNAD), that spe- recognition can occur and are considered to be of the “secondcifically converts NH,terminal asparagine residues of ary” or “tertiary“ destabilizing type
Ing that the enzyme can act well on substrates methionine residuesfrom proteins withMet-Gln would require with ionized or unionized a-amino groupsT. he proper- an aminopeptidase in contrastto the removal of Ac-Met, which ties and specificity of PNAD are consistent with those can be catalyzed by the well-characterized acylaminoacid hyexpected forthe enzyme required forthe ubiquitin-de- drolase
Conversion of the resulting In this report, we describe the identification, isolation, and NH,terminalasparagine to asparticacid byPNAD would render the protein susceptible to arginylation, polyubiquitinylationanddegradation as specified by the N-end rule
Summary
He proper- an aminopeptidase in contrastto the removal of Ac-Met, which ties and specificity of PNAD are consistent with those can be catalyzed by the well-characterized acylaminoacid hyexpected forthe enzyme required forthe ubiquitin-de- drolase Such proteins shouldNb“e-acetylatedon to aspartic acid would allow proteins with the initialsequence the retained initiator methionine and can subsequently ofAc-Met-Asn- to be recognized and degradedby the E3a pathbe modified by the removal of N-acetyl methionine by way following removal of the substituted methionine(Fig. 1). Ice-cold 5 M NH, acetate, pH7.0 (10pl), was added to neutralize the reaction mixture, and the samples were equilibrated onice for 10
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