Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.
Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.
Merola
M
M
Rockefeller University, New York, New York 10021.
Martínez del Pozo
A
A
Ueno
H
H
Recsei
P
P
Di Donato
A
A
Manning
J M
JM
Tanizawa
K
K
Masu
Y
Y
Asano
S
S
Tanaka
H
H
D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D051263:D-Alanine Transaminase; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005998:Glycine; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D012694:Serine; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D012694:Serine; D004228:Dithionitrobenzoic Acid; D000637:Transaminases; D051263:D-Alanine Transaminase; D003545:Cysteine; D005998:Glycine