Related Experiment Videos
Evidence against a covalent intermediate in the adenosine triphosphate phosphoribosyltransferase reaction of
Abstract:
14C-Labeled 5-phospho-alpha-D-ribose-1-diphosphate (PRibPP) was synthesized and its interaction with adenosine triphosphate phosphoribosyltransferase was examined by gel filtration in a search for a form of this substrate covalently bound to the enzyme. Wide variation in solvent conditions gave little labeling of the enzyme. Heavy labeling was found only in the presence of the second substrate, ATP, and this was shown to arise from tightly but noncovalently bound product. Previous reports of a covalent intermediate in this enzymatic reaction probably were due to contaminating ATP in 5-phospho-alpha-D-ribose-1-diphosphate. Feedback inhibition of the enzyme by histidine was shown to occur at the step giving product or at some earlier step in the mechanism.
Insights
Researchers investigated the enzyme adenosine triphosphate phosphoribosyltransferase using 14C-Labeled 5-phospho-alpha-D-ribose-1-diphosphate (PRibPP). They found no evidence of a covalent intermediate, suggesting previous reports were due to ATP contamination.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Adenosine triphosphate phosphoribosyltransferase (ATPR) is crucial in purine biosynthesis.
- Understanding its reaction mechanism, including potential intermediates, is vital for metabolic pathway elucidation.
Purpose of the Study:
- To investigate the interaction of 5-phospho-alpha-D-ribose-1-diphosphate (PRibPP) with ATPR.
- To determine if a covalent intermediate is formed during the enzymatic reaction.
- To clarify the mechanism of feedback inhibition by histidine.
Main Methods:
- Synthesis of 14C-labeled PRibPP.
- Gel filtration chromatography to assess enzyme-substrate interactions.
- Varying solvent conditions to optimize labeling.
- Analysis of product binding in the presence of ATP.
Main Results:
- No significant covalent labeling of ATPR with PRibPP was observed under various conditions.
- Heavy labeling occurred only in the presence of ATP, indicating tightly bound, noncovalent product.
- Contamination of PRibPP with ATP likely caused previous reports of a covalent intermediate.
- Histidine feedback inhibition occurs at or before product formation.
Conclusions:
- The reaction catalyzed by ATPR does not involve a covalent intermediate.
- Product inhibition is noncovalent and requires ATP.
- Histidine feedback inhibition affects an early step in the reaction mechanism.