Sulfuryl transfer catalyzed by pyruvate kinase

Area of Science:

• Biochemistry
• Enzymology
• Organic Synthesis

Background:

• Phosphoenolpyruvate (PEP) is a key intermediate in glycolysis and gluconeogenesis.
• Enzymes utilizing PEP play crucial roles in cellular metabolism.
• Understanding substrate analogues can elucidate enzyme mechanisms and allosteric regulation.

Purpose of the Study:

• To synthesize sulfoenolpyruvate (SEP), a sulfate ester analogue of PEP.
• To investigate the substrate specificity and kinetic properties of pyruvate kinase (PK) and phosphoenolpyruvate carboxylase (PEPC) with SEP.
• To characterize the interaction of SEP with PK and its allosteric regulation.

Main Methods:

• Chemical synthesis of SEP from ethyl bromopyruvate.
• Enzymatic assays using PK and PEPC.
• Product identification via NMR spectroscopy and comparison with authentic samples.
• Kinetic analysis of enzyme-substrate interactions.

Main Results:

• SEP was synthesized in 40% overall yield.
• SEP is a substrate for PK, yielding pyruvate and adenosine 5'-sulfatopyrophosphate (SAPP).
• Sulfuryl transfer from SEP is 250-600 times slower than phosphate transfer from PEP.
• SEP is not a substrate for PEPC.
• SEP binds to a site distinct from the active site of PK, activating the enzyme similarly to glucose 6-phosphate.

Conclusions:

• SEP is a viable substrate for pyruvate kinase, albeit with reduced catalytic efficiency.
• The distinct binding and activation mechanism of SEP highlights allosteric regulation in PK.
• SEP serves as a valuable tool for probing the active site and regulatory mechanisms of PEP-utilizing enzymes.