Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase.
Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.
Hernandez
D
D
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16803.
Stroh
J G
JG
Phillips
A T
AT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D006638:Histidine Ammonia-Lyase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D016958:Pseudomonas putida / Q000201:enzymology*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine
D010446:Peptide Fragments; D012694:Serine; D006638:Histidine Ammonia-Lyase