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Evidence for proximal cysteine and lysine residues at or near the ative site of arginine kinase of Stichopus japonicus

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Abstract

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.

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Abbreviations

FPLC:

fast performance liquid chromatography

AK:

arginine kinase

CD:

circular dichroism

CK:

creatine kinase

DTNB:

5,5′-dithiobis(2-nitrobenzoic acid)

OPTA:

o-phthalaldehyde

PArg:

phosphoarginine

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Authors and Affiliations

  1. Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, 100084, Beijing, China

    Qin Guo, BaoYu Chen & XiCheng Wang

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  1. Qin Guo
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  2. BaoYu Chen
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  3. XiCheng Wang
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Correspondence to XiCheng Wang.

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Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1639–1648.

Original Russian Text Copyright © 2004 by Qin Guo, BaoYu Chen, XiCheng Wang.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-047, June 27, 2004.

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Guo, Q., Chen, B. & Wang, X. Evidence for proximal cysteine and lysine residues at or near the ative site of arginine kinase of Stichopus japonicus . Biochemistry (Moscow) 69, 1336–1343 (2004). https://doi.org/10.1007/s10541-005-0078-3

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  • DOI: https://doi.org/10.1007/s10541-005-0078-3

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