Structural and catalytic role of arginine 88 in Escherichia coli adenylate kinase as evidenced by chemical modification and site-directed mutagenesis.
Phenylglyoxal inactivates Escherichia coli adenylate kinase by modifying a single arginine residue (Arg-88). ATP, ADP, P1,P5-di(adenosine 5')-pentaphosphate, and to a lesser extent AMP protect the enzyme against inactivation by phenylglyoxal. Site-directed mutagenesis of Arg-88 to glycine yields a modified form of adenylate kinase (RG88 mutant) closely related structurally to the wild-type protein as indicated by Fourier transform infrared spectroscopy, differential scanning calorimetry, and limited proteolysis. However, this modified protein has only 1% of the maximum catalytic activity of the wild-type enzyme and 5- and 85-fold higher apparent Km values for ATP and AMP, respectively, than the parent adenylate kinase. Arg-88, which is a highly conserved residue in all known molecular forms of adenylate kinases (corresponding to Arg-97 in muscle cytosolic enzyme), should be located inside a big cleft of the molecule, close to the phosphate-binding loop. It possibly stabilizes the transferable gamma-phosphate group from ATP to AMP in the transition state.
Reinstein
J
J
Max-Planck Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, West Germany.
Gilles
A M
AM
Rose
T
T
Wittinghofer
A
A
Saint Girons
I
I
Bârzu
O
O
Surewicz
W K
WK
Mantsch
H H
HH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D001120:Arginine; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005583:Fourier Analysis; D006358:Hot Temperature; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism; D009154:Mutation; D010658:Phenylglyoxal / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D015226:Dinucleoside Phosphates; D000249:Adenosine Monophosphate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases; D000263:Adenylate Kinase; D003012:Microbial Collagenase; D010658:Phenylglyoxal