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 fr om Escherichia coli was irreversibly inactivated on exposure to the af finity analog 2',3'-dialdehyde ATP (oATP). The reaction displayed comp lex saturation kinetics with respect to oATP with an apparent K-D of a pproximately 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 c ould be protected against inactivation by ADP or ATP. Isolation of rad ioactive peptides from the enzyme modified with radioactive oATP, foll owed by automated Edman sequencing allowed identification of Lys(181), Lys(193), and Lys(230) as probable sites of reaction with the analog. Cysteine 229 may also be labeled by oATP. Of these four residues, Lys (193) is completely conserved within the family of PRPP synthetases, a nd Lys(181) is found at a position in the sequence where the cognate a mino acid (Asp(181)) in human isozyme I PRPP synthetase has been previ ously implicated in the regulation of enzymatic activity. These result s imply a functional role for at least two of the identified amino aci d residues.