Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases.
NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyze opposing arms of a putative ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian tissues and Rhodospirillum rubrum exhibit three regions of similarity in deduced amino acid sequence. We postulated that amino acids in these consensus regions could be critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Conserved amino acids in each of these regions were altered by site-directed mutagenesis. Replacement of Asp-60 or Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme activity. The double Asp-60 --> Glu/Asp-61 --> Glu mutant was inactive, as were Asp-60 --> Gln/Asp-61 --> Gln or Asp-60 --> Asn/Asp-61 --> Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65, Arg-139, Asp-285, which are also located in the conserved regions, with alanine did not change specific activity. These data clearly show that the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine hydrolase are critical for catalytic activity, but not for high affinity binding of the substrate analogue, ADP-ribose.
Konczalik
P
P
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434, USA. konczalp@gwgate.nhlbi.nih.gov
Moss
J
J
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology; D013045:Species Specificity
D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase