Evidence for Proximal Cysteine and Lysine Residues at or near the Active Site of Arginine Kinase of Stichopus japonicus

---

Qin Guo, BaoYu Chen, and XiCheng Wang^*

Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084 China; fax: 8610-62784977; E-mail: wangxic@mail.tsinghua.edu.cn

^* To whom correspondence should be addressed.

Received February 26, 2004; Revision received April 21, 2004

---

Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou's method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5´-dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274.

---

KEY WORDS: arginine kinase, cysteine, OPTA, Tsou's method

---