Mammalian glycinamide ribonucleotide transformylase: purification and some properties

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• De novo purine biosynthesis is essential for DNA and RNA synthesis.
• Glycinamide ribonucleotide transformylase (GAR transformylase) is a key enzyme in this pathway, utilizing 10-formyltetrahydrofolate.
• Understanding GAR transformylase's properties is vital for comprehending purine metabolism.

Purpose of the Study:

• To purify and characterize the mammalian GAR transformylase.
• To investigate the enzyme's kinetic properties and reaction mechanism.
• To provide insights into the regulation of de novo purine biosynthesis.

Main Methods:

• Purification of GAR transformylase from murine lymphoma cell line L5178Y using a gelatin protease affinity resin.
• Determination of enzyme homogeneity and molecular weight (Mr ~110,000).
• Kinetic studies to determine Michaelis constants (Km) and assess the reaction mechanism.

Main Results:

• Achieved a 1500-fold purification of GAR transformylase to near homogeneity.
• Identified the enzyme as a monomeric protein with an Mr of approximately 110,000.
• Kinetic analyses indicated a sequential reaction mechanism with specific Km values for glycinamide ribonucleotide (0.4 mM) and 10-formyl-5,8-dideazafolate (0.25 µM).
• Determined a minimum Vmax of 2 µmol/(min·mg) and a turnover number of 4 s⁻¹.

Conclusions:

• The mammalian GAR transformylase has been successfully purified and characterized.
• The enzyme exhibits kinetic properties consistent with a sequential reaction mechanism.
• These findings contribute to a deeper understanding of the de novo purine biosynthesis pathway and its regulation.