Histidine ammonia-lyase mutant S143C is posttranslationally converted into fully active wild-type enzyme. Evidence for serine 143 to be the precursor of active site dehydroalanine.
Histidase [histidine ammonia-lyase (HAL); EC 4.3.1.3] from Pseudomonas putida is a homotetramer and contains one catalytically essential dehydroalanine residue per subunit. Since the mutant S143A was catalytically inert, it has been proposed that serine 143 is the precursor of the active site dehydroalanine [Langer et al. (1994) Biochemistry 33, 6462-6467]. To further define the role of serine 143, we prepared the mutants S143T and S143C by site-directed mutagenesis. The threonine 143 mutant was neither catalytically active (< 0.01%) nor did it form with L-cysteine and oxygen a product absorbing at 340 nm. In contrast, the cysteine 143 mutant showed full catalytic activity and, after treatment with L-cysteine and oxygen, an increased absorbance at 340 nm similar to that of the wild-type enzyme. Also the kinetic constants (Km and Vmax) were identical with those of wild-type histidase. Titration with Ellman's reagent revealed that both wild-type and S143C mutant histidase contained seven thiol groups after exhaustive reduction. It must be concluded that posttranslational modification occurs with both serine 143 and cysteine 143 by elimination of water and hydrogen sulfide, respectively. In both cases dehydroalanine is formed and the resulting histidases are indistinguishable. In contrast, the threonine 143 mutant is not processed to active enzyme.
Langer
M
M
Department of Biochemistry, University of Karlsruhe, Germany.
Lieber
A
A
Rétey
J
J
D000409:Alanine / Q000031:analogs & derivatives*; D001483:Base Sequence; D003545:Cysteine / Q000494:pharmacology; D006638:Histidine Ammonia-Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000494:pharmacology; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016958:Pseudomonas putida / Q000201:enzymology; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis; D013912:Threonine
D011498:Protein Precursors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013912:Threonine; D012694:Serine; C015102:dehydroalanine; D006638:Histidine Ammonia-Lyase; D003545:Cysteine; D000409:Alanine; D010100:Oxygen