Ser-143 is an essential active site residue in histidine ammonia-lyase of Pseudomonas putida.
Site directed mutagenesis was used to investigate the role of Ser-143 in enzyme activity and as a point for attack by cyanide or L-cysteine, two irreversible inhibitors of histidine ammonia-lyase (histidase). Two mutant proteins, a S143A substitution and an A142S-S143A transposition, were made. Both mutant histidases completely lost all enzymatic activity. Western blots with anti-histidase antibodies revealed that the mutant proteins were being expressed at a level equal to that of the wild-type protein. The purified mutant proteins could not incorporate [14C]cyanide nor could they generate the UV-absorbing species normally observed when L-cysteine modifies wild-type histidase. These results support the hypothesis that Ser-143 is the binding site for an as yet unidentified histidase cofactor.
Hernandez
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Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.
Phillips
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006638:Histidine Ammonia-Lyase / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D006638:Histidine Ammonia-Lyase