Two Fe2+ atoms are present in activated prolyl 4-hydroxylase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen synthesis.
• The enzyme's activity is known to be influenced by metal cofactors.

Purpose of the Study:

• To elucidate the relationship between prolyl 4-hydroxylase activity and Fe2+ binding.
• To determine the stoichiometry of Fe2+ binding to the enzyme.

Main Methods:

• Enzyme kinetics assays were performed.
• Quantification of bound Fe2+ was conducted at varying enzyme activities.

Main Results:

• A linear correlation was established between prolyl 4-hydroxylase activity and the molar amount of bound Fe2+.
• At maximal activity, the enzyme tetramer was found to bind 2.1 mol of Fe2+ per mol, indicating two specific Fe2+-binding sites.
• The concentration of Fe2+ required for half-maximal enzyme activity was dependent on the type of sulphydryl compounds present.

Conclusions:

• Fe2+ is a critical cofactor for prolyl 4-hydroxylase, with two binding sites per tetramer.
• The enzyme's catalytic efficiency is directly proportional to its Fe2+ saturation.
• Reaction conditions, specifically sulphydryl compounds, modulate the enzyme's affinity for Fe2+.