Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP ( or GTP) and AMP to generate ADP ( or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol(-1). The K-m of the CsAK3 for AMP and GTP are 118 mu M and 359 mu M, respectively. CsAK3 is inhibited by Ap5A (> 70% inhibition by 2.0 mM AP(5)A). Ap(5)A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.