Abstract
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.
Highlights
From The Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen K, Denmark
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
Our affinity labeling results localize the sites of reaction of oATP with PRPP synthetase to a region of sequence spanning approximately 60 amino acid residues
Summary
Affinity labeling studies with 5'-p-fluorosulfonylbenzoyladenosine [19], mutagenesis studies [20], and characterization of mutants isolated by genetic methods [21] have led to the identification of a region of highly conserved amino acid sequence within the PRPP synthetase family This region may be involved in ATP or divalent cation binding and includes a histidyl residue that may act as a general base during catalysis. The affinity analog prepared by treating ATP with sodium periodate, oATP [25], presents an attractive opportunity to identify this putative functionallysyl residue because the reagent contains the same reactive moiety as pyridoxal-5-phos-
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