Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site.

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.

Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium.

The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to two rat isozyme sequences. Strains containing plasmid-borne prsA have been used to overproduce and purify the enzyme. The promoter for the S. typhimurium prsA gene was identified by deletion analysis and by similarity to the promoter for the E. coli prsA gene. The location of the prsA promoter results in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional significance.

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.

Mechanism of inactivation of glutamine amidotransferases by the antitumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125).

L-(alphaS, 5S)-alpha-Amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an antitumor drug isolated from Streptomyces sviceus, is an active site-directed affinity analog of glutamine. It selectively inactivates the glutamine-dependent activities of two bacterial glutamine amidotransferases, anthranilate synthase and glutamate synthase. A reversible noncovalent complex is formed prior to irreversible enzyme modification. Inactivation of anthranilate synthase results from incorporation of approximately 1 eq of AT-125/enzyme protomer. Active site cysteine-83 in Serratia marcescens anthranilate synthase Component II is the residue alkylated by AT-125. Anthranilate synthase is rapidly inactivated by AT-125 IN S. marcescens cells. In vivo inactivation is by the same mechanism as in vitro.