Chemical modification and active site studies of Salmonella typhimurium phosphoribosylpyrophosphate synthetase

Comparatively little is known about the amino acid residues present at the active site of Salmonella typhimurium phosphoribosylpyrophosphate synthetase (E.C. 2.7.6.1, PRPP synthetase). Chemical modification studies using group specific and affinity labelling reagents were used to study the active site residues of the enzyme. Peptide mapping of the enzyme was performed to provide a method for rapidly locating and identifying the sites of reaction. CNBr and tryptic maps were prepared using reverse phase HPLC. Peptides from these maps were isolated and sequenced. About 85% of the primary amino acid sequence was covered. Previous work (Roberts et al., J. Biol. Chem, (1975) 250:5364-5369) suggested the presence of an essential cysteinyl residue in the active site. The present results do not support this conclusion. PRPP synthetase contains four cysteinyl residues. A single, reactive cysteinyl residue, Cys$\sb{229}$, was identified using radioactive iodoacetamide. The other 3 cysteinyl residues were equivalent in reactivity, and were labeled with iodoacetamide only under denaturing conditions. With 5,5$\sp\prime$-dithiobis(nitrobenzoic acid) reactivity is similar, except that the 3 less reactive residues react slowly under nondenaturing conditions. The extent of reaction with DTNB is dependent on the concentration of inorganic phosphate. When all the cysteinyl residues have reacted with DTNB the enzyme still possessed about 20% of the control enzymatic activity.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Harlow, Kenneth William

• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1989 Harlow, Kenneth William

Language dc:language

eng

Identifier

AAI8924830
(UMI)AAI8924830

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21814