Abstract
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.
Highlights
From the Department of Molecular and Medical Genetics, University of Toronto, and Department of Genetics, Research Institute, The Hospital for Sick Children, lbronto, Ontario M5G 1x8,Canada
We have identified serin25e4 as an essential residue in Formation of the dehydroalanine residue in histidase and rat histidase
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 cDNACsOSincells, which sistent with the generatioofndehydroalanine by autocatalysis, since it is less likely that modifying enzymes for dehydroalanine formation are present in thesecells
Summary
Site-directed Mutagenesisof Conserved Serines in Rat Histidase We have identified serin25e4 as an essential residue in Formation of the dehydroalanine residue in histidase and rat histidase (histidine ammonia-lyasEe,C 4.3.1.3). FIG1..Alignment of deduced amino acid sequencesfrom four histidine ammonia-lyases( H A L )and 10 phenylalanine ammonialyases (PAL).Sequence alignment was performedas described under "ExperimentPalrocedures." The 46 identicarlesidues areboxed, and the four serines mutated in this study (positions 223,254,508, and 53H3ALin) araet indicated bydots.
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