Inactivation of Escherichia coli phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP. Identification of active site lysines.
The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2',3'-dialdehyde ATP (oATP). The reaction displayed complex saturation kinetics with respect to oATP with an apparent KD of approximately 0.8 mM. Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit. oATP served as a substrate in the presence of ribose-5-phosphate, and the enzyme could be protected against inactivation by ADP or ATP. Isolation of radioactive peptides from the enzyme modified with radioactive oATP, followed by automated Edman sequencing allowed identification of Lys181, Lys193, and Lys230 as probable sites of reaction with the analog. Cysteine 229 may also be labeled by oATP. Of these four residues, Lys193 is completely conserved within the family of PRPP synthetases, and Lys181 is found at a position in the sequence where the cognate amino acid (Asp181) in human isozyme I PRPP synthetase has been previously implicated in the regulation of enzymatic activity. These results imply a functional role for at least two of the identified amino acid residues.
Hilden
I
I
Center for Enzyme Research, University of Copenhagen, Denmark.
Hove-Jensen
B
B
Harlow
K W
KW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D000345:Affinity Labels; D010446:Peptide Fragments; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D012268:Ribose-Phosphate Pyrophosphokinase; D014357:Trypsin; D008239:Lysine