Sulfhydryl chemistry of Salmonella typhimurium phosphoribosylpyrophosphate synthetase: identification of two classes of cysteinyl residues.
Sulfhydryl-specific reagents were used to study the reactivities and function of the four cysteinyl residues per subunit present in Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. In the presence of high concentrations of denaturants all four cysteinyl residues reacted with sulfhydryl-specific reagents. In the absence or in the presence of low levels of denaturing agents, two classes of cysteinyl residues were identified. A single sulfhydryl reacted rapidly with iodoacetamide and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) without significant loss of enzymatic activity. This single sulfhydryl was identified as Cys-229 by reaction with iodo[1-14C]acetamide, followed by isolation and sequence analysis of a single radiolabeled peptide. The three remaining sulfhydryls reacted to various extents depending on the conditions and sulfhydryl-specific reagents employed. At low Pi concentrations, these residues reacted fully with DTNB, leading to an 80 to 90% loss of enzymatic activity. ATP and high levels of Pi prevented this reaction. These results, along with studies comparing the S. typhimurium PRPP synthetase sequence with the sequences of PRPP synthetases from other species, suggest that the cysteinyl residues in the Salmonella enzyme are not catalytically essential. That one or more of the three less reactive residues may lie in or near the active site is not excluded.
Harlow
K W
KW
Department of Biochemistry, University of Illinois, Urbana 61801.
Switzer
R L
RL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000378:metabolism*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase; D003545:Cysteine; D003488:Cyanogen Bromide; D007460:Iodoacetamide