EVIDENCE FOR PARTICIPATION OF ASPARTATE-84 AS A CATALYTIC GROUP AT THE ACTIVE-SITE OF PORPHOBILINOGEN DEAMINASE OBTAINED BY SITE-DIRECTED MUTAGENESIS OF THE HEMC GENE FROM ESCHERICHIA-COLI

The role of aspartate-84, an invariant residue in the active site clef t of Escherichia coil porphobilinogen deaminase, has been investigated by site-directed mutagenesis. Substitution of aspartate-84 by glutama te results in an enzyme that retains less than 1% of its activity and which can form highly stable enzyme-intermediate complexes. Substituti on of aspartate-84 by either alanine or asparagine, however, results i n proteins unable to catalyze the formation of preuroporphyrinogen but which, nevertheless, appear able to assemble the dipyrromethane cofac tor. The mechanisms of the tetramerization reaction and cofactor assem bly are discussed.