Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase

Area of Science:

• Biochemistry
• Enzymology
• Vitamin B12 Metabolism

Background:

• Adenosylcobalamin (AdoCbl) is a crucial coenzyme in various metabolic reactions.
• Understanding the structural requirements for AdoCbl's coenzymic activity is vital.

Purpose of the Study:

• To synthesize and characterize a novel AdoCbl analog, adenosylcobinamide methyl phosphate, lacking the nucleotide loop.
• To investigate the role of the nucleotide loop in AdoCbl binding, activation, and catalysis.

Main Methods:

• Chemical synthesis of adenosylcobinamide methyl phosphate.
• Enzyme kinetic studies to assess inhibitory and coenzymic activity.
• Incubation of apoenzyme with the analog and substrate, followed by analysis of reaction products and enzyme modification.

Main Results:

• The analog exhibited competitive inhibition of AdoCbl with a Ki of 2.5 microM.
• In the presence of substrate, the analog's Co-C bond was irreversibly cleaved stoichiometrically, forming an enzyme-bound Co(II) species.
• 5'-Deoxyadenosine was the sole product from the adenosyl moiety, and the apoenzyme remained unmodified.
• Adenosylcobinamide alone did not bind tightly or function as a coenzyme.

Conclusions:

• Adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or suicide coenzyme.
• The phosphodiester moiety of the nucleotide loop is essential for tight apoenzyme binding, Co-C bond activation, and catalysis.
• The nucleotide loop is obligatory for the normal catalytic cycle of AdoCbl-dependent enzymes.