Chemical modification and active site studies of Salmonella typhimurium phosphoribosylpyrophosphate synthetase
Harlow, Kenneth William
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https://hdl.handle.net/2142/21814
Description
Title
Chemical modification and active site studies of Salmonella typhimurium phosphoribosylpyrophosphate synthetase
Author(s)
Harlow, Kenneth William
Issue Date
1989
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
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.
The ATP affinity analog 5$\sp\prime$-($p$-fluorosulfonylbenzoyl)adenosine (FSBA) was used to covalently label the enzyme. It reacted with the enzyme in a limited, active site directed manner, with an apparent K$\sb{\rm D}$ of 1.7 mM. The modification was labile at alkaline and acidic pH, and no acid-stable products of reaction of FSBA with amino acid residues in proteins were identified. The site of reaction was localized to a 9 residue peptide (residues 124-132) with histidine (His$\sb{130}$) as the only potential site of reaction with FSBA. His$\sb{130}$ is invariably conserved in PRPP synthetases from diverse origins. These results are consistent with His$\sb{130}$ being the site of reaction of FSBA with PRPP synthetase, and with His$\sb{130}$ being important to the functioning of the enzyme.
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