Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification.
Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.
Nakano
Y
Y
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Tokyo.
Itoh
M
M
Tanaka
E
E
Kimura
K
K
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D044943:Fatty Acid Desaturases; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors
D009282:Naphthalenesulfonates; D008345:Manganese; C006421:1,5-I-AEDANS; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium; D000241:Adenosine; D003545:Cysteine