S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization

Area of Science:

• Biochemistry
• Enzymology
• Protein Purification

Background:

• S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) is a key enzyme in cellular metabolism.
• Understanding its structure and function is crucial for comprehending metabolic regulation.

Purpose of the Study:

• To purify and characterize S-Adenosylhomocysteine hydrolase from human placenta.
• To investigate the enzyme's quaternary structure, cofactor binding, and substrate interactions.

Main Methods:

• Purification to homogeneity using S-adenosylhomocysteine-agarose affinity chromatography.
• Determination of molecular weight, subunit composition, and isoelectric point.
• High-performance liquid chromatography (H.p.l.c.) for cofactor and substrate binding analysis.

Main Results:

• The enzyme is a tetramer (Mr 189,000) with subunits of Mr 47,000-48,000, containing nine cysteine residues per subunit and no disulfide bonds.
• Four molecules of NAD cofactor are tightly bound per tetramer, with 10-50% in the reduced form; four adenosine binding sites exist per tetramer.
• The enzyme catalyzes the interconversion of adenosine and 4',5'-dehydroadenosine, with an equilibrium favoring the latter; enzyme activity correlates with the NAD+/NADH ratio.

Conclusions:

• Human placental S-Adenosylhomocysteine hydrolase is a tetrameric enzyme with tightly bound NAD.
• Enzyme activity and adenosine binding are influenced by the redox state of the bound NAD and the enzyme's adenosine content.
• The enzyme's sensitivity to adenosine inactivation is dependent on the presence of dithiothreitol.