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Active site lysines in orotate phosphoribosyltransferase
C Grubmeyer1, E Segura, R Dorfman
1Department of Biochemistry, Temple University Medical School, Philadelphia, Pennsylvania 19140.
The Journal of Biological Chemistry
|September 25, 1993
Summary
Orotate phosphoribosyltransferase (OPRTase) enzyme structure was studied using chemical modification. Key lysine residues in the active site were identified, revealing their roles in binding substrates like orotate and PRPP.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Orotate phosphoribosyltransferase (OPRTase) is crucial for nucleotide biosynthesis.
- Bacterial OPRTase is a monofunctional dimer, distinct from its mammalian counterpart.
- Understanding OPRTase structure-function relationships is vital for drug development.
Purpose of the Study:
- To elucidate the role of basic residues in the active site of Salmonella typhimurium OPRTase.
- To identify specific lysine residues involved in substrate binding and catalysis.
- To map the active site of OPRTase through chemical modification and sequence analysis.
Main Methods:
- Purification of crystalline Salmonella typhimurium OPRTase.
- Chemical modification using 2,4,6-trinitrobenzene sulfonate (TNBS).
- Enzyme inactivation kinetics, spectral analysis, tryptic proteolysis, HPLC, and amino acid sequencing.
Main Results:
- TNBS inactivated OPRTase by modifying three key lysine residues (Lys-26, Lys-100, Lys-103).
- Substrates orotidine-5'-monophosphate (OMP) and 5-phosphoribosyl-1-pyrophosphate (PRPP) protected these lysines from modification.
- Lys-26 interacts with the ribose-phosphate moiety, while Lys-100 and Lys-103 interact with the pyrophosphate moiety of PRPP.
Conclusions:
- Identified active site lysine residues crucial for OPRTase function.
- Provided insights into the catalytic mechanism and substrate binding of OPRTase.
- Structural information can guide the design of OPRTase inhibitors.