Site-directed mutagenesis of conserved serines in rat histidase. Identification of serine 254 as an essential active site residue.
We have identified serine 254 as an essential residue in rat histidase (histidine ammonia-lyase, EC 4.3.1.3). Histidase and phenylalanine ammonia-lyase are the only two enzymes that have been postulated to require the modified amino acid, dehydroalanine, for enzyme activity. In the bacterial peptides nisin and subtilin, and in the pyruvoyl enzymes, the precursor for dehydroalanine is a serine. To determine whether serine may be the dehydroalanine precursor in rat histidase, we substituted four highly conserved serines with alanines, and expressed the mutated histidase cDNAs in COS cells, which have no endogenous histidase activity. Substitution of serines 223, 254, 508, and 533 resulted in the production of approximately equal amounts of histidase protein with histidase activities of 2.4, 0.0, 75, and 16%, respectively. The abrogation of histidase activity by the substitution of alanine for serine 254, together with the modification by L-cysteine of the corresponding residue in Pseudomonas putida histidase (Hernandez, C., Stroh, J. G., and Phillips, A. T. (1993) Arch. Biochem. Biophys. 307, 126-132), is strong evidence that this residue is the precursor of the essential electrophilic moiety of histidase.
Taylor
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RG
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
McInnes
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D006638:Histidine Ammonia-Lyase / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014162:Transfection
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012694:Serine; D010650:Phenylalanine Ammonia-Lyase; D006638:Histidine Ammonia-Lyase