HISTIDINE AMMONIA-LYASE MUTANT-S143C IS POSTTRANSLATIONALLY CONVERTEDINTO FULLY ACTIVE WILD-TYPE ENZYME - EVIDENCE FOR SERINE-143 TO BE THE PRECURSOR OF ACTIVE-SITE DEHYDROALANINE
M. Langer et al., HISTIDINE AMMONIA-LYASE MUTANT-S143C IS POSTTRANSLATIONALLY CONVERTEDINTO FULLY ACTIVE WILD-TYPE ENZYME - EVIDENCE FOR SERINE-143 TO BE THE PRECURSOR OF ACTIVE-SITE DEHYDROALANINE, Biochemistry, 33(47), 1994, pp. 14034-14038
Histidase [histidine ammonia-lyase (HAL); EC 4.3.1.3] from Pseudomonas
putida is a homotetramer and contains one catalytically essential deh
ydroalanine residue per subunit. Since the mutant S143A was catalytica
lly inert, it has been proposed that serine 143 is the precursor of th
e active site dehydroalanine [Langer et al. (1994) Biochemistry 33, 64
62-6467]. To further define the role of serine 143, we prepared the mu
tants 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 treatmen
t with L-cysteine and oxygen, an increased absorbance at 340 nm simila
r to that of the wild-type enzyme. Also the kinetic constants (K-m and
V-max) were identical with those of wild-type histidase. Titration wi
th Ellman's reagent revealed that both wild-type and S143C mutant hist
idase contained seven thiol groups after exhaustive reduction. It must
be concluded that posttranslational modification occurs with both ser
ine 143 and cysteine 143 by elimination of water and hydrogen sulfide,
respectively. In both cases dehydroalanine is formed and the resultin
g histidases are indistinguishable. In contrast, the threonine 143 mut
ant is not processed to active enzyme.