Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida.
Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
D000409:Alanine / Q000031:analogs & derivatives* / Q000032:analysis; D000642:Ammonia-Lyases / Q000032:analysis*; D001665:Binding Sites; D002384:Catalysis; D003486:Cyanides / Q000494:pharmacology; D004120:Dimethyl Suberimidate; D006638:Histidine Ammonia-Lyase / Q000032:analysis*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011549:Pseudomonas / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D003486:Cyanides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D004120:Dimethyl Suberimidate; D012694:Serine; C015102:dehydroalanine; D000642:Ammonia-Lyases; D006638:Histidine Ammonia-Lyase; D000409:Alanine