Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism

• Published in: International journal of biological macromolecules 2017-08, Vol.101, p.653-659
• Main Authors: Yano, Daichi, Suzuki, Takaya, Hirokawa, Saki, Fuke, Kyoko, Suzuki, Tomohiko
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arginine kinase; Arginine Kinase - antagonists & inhibitors; Arginine Kinase - chemistry; Arginine Kinase - genetics; Arginine Kinase - metabolism; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Paramecium tetraurelia; Paramecium tetraurelia - enzymology; Phosphagen kinase; Prenylation; Protein Conformation; RNA, Messenger - genetics; RNA, Messenger - metabolism; Substrate inhibition
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2017.03.133

One of the Paramecium arginine kinases shows typical substrate inhibition toward arginine, and the Cys residue in C-terminal region of the enzyme is farnesylated. The residue S79 is responsible for the substrate inhibition. [Display omitted] •Four AKs from the ciliate Paramecium tetraurelia have been characterized.•AK3 showed substrate inhibition toward arginine. Residue S79 is essential for this unusual phenomenon.•The extremely low enzyme activity of AK4 was attributed to the unusual replacement of G298 by R. The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1–3) via PCR. Recombinant AK1–4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77–81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.