Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase.
Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine. Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme. Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.
Gilles
A M
AM
Unité de Chimie des Protéines, Institut Pasteur, Paris, France.
Marlière
P
P
Rose
T
T
Sarfati
R
R
Longin
R
R
Meier
A
A
Fermandjian
S
S
Monnot
M
M
Cohen
G N
GN
Bârzu
O
O
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000263:Adenylate Kinase / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis; D000614:Aminocaproates; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine; D009646:Norleucine; D010770:Phosphotransferases / Q000032:analysis*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000614:Aminocaproates; D000249:Adenosine Monophosphate; D009646:Norleucine; D008715:Methionine; D006861:Hydrogen Peroxide; D010770:Phosphotransferases; D000263:Adenylate Kinase; D003488:Cyanogen Bromide