Substrate share in the suicide inactivation of mushroom tyrosinase Kamahldin Haghbeen a, * , Ali Akbar Saboury b , Farhad Karbassi b a The National Research Institute for Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran b Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran Received 28 June 2004; received in revised form 12 August 2004; accepted 30 August 2004 Available online 17 September 2004 Abstract To address the real cause of the suicide inactivation of mushroom tyrosinase (MT), under in vitro conditions, cresolase and catecholase reactions of this enzyme were investigated in the presence of three different pairs of substrates, which had been selected for their structural specifications. It was showed that the cresolase activity is more vulnerable to the inactivation. Acetylation of the free tyrosyl residues of MT did not cure susceptibility of the cresolase activity, but clearly decreased the inactivation rate of MT in the presence of 4-[(4- methylbenzo)azo]-1,2-benzenediol (MeBACat) as a catecholase substrate. Considering the results of the previous works and this research, some different possible reasons for the suicide inactivation of MT have been discussed. Accordingly, it was proposed that the interruption in the conformational changes in the tertiary and quaternary structures of MT, triggered by the substrate then mediated by the solvent molecules, might be the real reason for the suicide inactivation of the enzyme. However, minor causes like the toxic effect of the ortho -quinones on the protein body of the enzyme or the oxidation of some free tyrosyl residues on the surface of the enzyme by itself, which could boost the inactivation rate, should not be ignored. D 2004 Elsevier B.V. All rights reserved. Keywords: Mushroom tyrosinase; Suicide inactivation; Substrate structure 1. Introduction Tyrosinase (EC 1.14.18.1) is able to carry out regiospe- cific hydroxylation on phenols and convert them to the ortho -dihydroxy compounds [1]. Although tyrosinase goes further and oxidizes the produced ortho -dihydroxy com- pound to the corresponding ortho -quinone; this last reaction could be offset by using an external reducing material like ascorbic acid or hydroxylamine [2]. Tyrosinase can be extracted from inexpensive sources like mushroom and mushroom tyrosinase (MT) has showed attractive properties [3]. Its active site seems to be close to the surface of the protein; therefore, it is accessible for the phenolic residues of big molecules like peptides [4]. Numerous researches have proved its widespread affinity for different substrates [5–9]. So, it is quite promising for industrial purposes. Besides, MT is a tetrameric polypeptide similar to the mammalian tyrosinase, the key enzyme in melanogenesis [10–12], and its clinical applications have been showed [13]. Yet, it is not economic to use MT in a large-scale plan due to its rather fast inactivation in the presence of its substrates [14]. Suicide inactivation of tyrosinases had been reported even in early works [15,16] and since then, many researchers have studied this bothering phenomenon from different angles [17–22] but not in a conclusive way. An enzyme commits suicide inactivation for different reasons [23], but considering previous works on MT and the aim of this research, five more important factors have been selected to be discussed in this paper: (1) The final product of the enzymatic reaction bonds to the enzyme and makes it incompetent. (2) There is a dead-end product for the reaction of the enzyme with its substrate. (3) As a result of the enzymatic reaction, a reactive molecule is produced which can assail the enzyme structure and knocks down it. (4) The enzyme attacks itself and brings about inefficiency. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.017 * Corresponding author. Tel.: +98 21 4580372; fax: +98 21 4580309. E-mail address: kamahl@nrcgeb.ac.ir (K. Haghbeen). Biochimica et Biophysica Acta 1675 (2004) 139 – 146 http://www.elsevier.com/locate/bba