Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose

doi:10.1016/j.ijbiomac.2011.12.035

International Journal of Biological Macromolecules

Volume 50, Issue 3, 1 April 2012, Pages 694-700

https://doi.org/10.1016/j.ijbiomac.2011.12.035 Get rights and content

Abstract

Tyrosinase plays a central role in biological pigment formation, and hence knowledge of tyrosinase catalytic mechanisms and regulation may have medical, cosmetic, and agricultural applications. We found in this study that arabinose significantly inhibited tyrosinase, and this was accompanied by conformational changes in enzyme structure. Kinetic analysis showed that arabinose-mediated inactivation followed first-order kinetics, and single and multiple classes of rate constants were measured. Arabinose displayed a mixed-type inhibitory mechanism with K_i = 0.22 ± 0.07 mM. Measurements of intrinsic and ANS-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. We simulated the docking between tyrosinase and arabinose (binding energies were −26.28 kcal/mol for Dock6.3 and −2.02 kcal/mol for AutoDock4.2) and results suggested that arabinose interacts mostly with His61, Asn260, and Met280. The present strategy of predicting tyrosinase inhibition by simulation of docking by hydroxyl groups may prove useful in screening for potential tyrosinase inhibitors, as shown here for arabinose.

Introduction

Tyrosinase (EC 1.14.18.1) is a ubiquitous enzyme with diverse physiological roles related to pigment production. Tyrosinase catalyzes the pigmentation of skin [1], [2], the browning of fruits and vegetables [3], [4], wound healing [5], [6], and cuticle formation in insects [7], [8]. Tyrosinase inhibitors have potential applications in medicine, cosmetics (e.g., as whitening agents), and agriculture (as bio-insecticides). Structurally, tyrosinase belongs to the type 3 copper protein family [9], [10], with two copper ions each coordinately bonded with a distinct set of three histidine residues within the active site [11]. The tyrosinase mechanism is complex, in that this enzyme can catalyze multiple reactions. The copper atoms participate directly in hydroxylation of monophenols to o-diphenols (cresolase activity) and in the oxidation of o-diphenols to o-quinones (catechol oxidase activity) [12]. The overall 3D structures and architecture of the active site in tyrosinase from various sources are gradually emerging [11], [13], [14]. Studies of this enzyme mechanism demand a variety of kinetic and computational methods to derive key structure–function relationships, e.g., between substrates and ligands of the enzyme [15], [16], [17], [18], [19].

Arabinose is a natural five-carbon sugar component of vegetables and fruits, an aldopentose having both sugar and aldehyde groups in its structure [20]. The inhibitory effects of some compounds with sugar backbones on tyrosinase may be of interest in studies of the enzyme mechanism [21], [22], [23], [24], [25]. The d-arabinose was used to immobilize mushroom tyrosinase on a re-usable glass bead preparation [26]. In this context, the binding affinities of arabinose for tyrosinase and copper ions at the tyrosinase active site identified arabinose as a potential tyrosinase inhibitor.

In this study, we investigated the mechanism of tyrosinase inhibition and unfolding induced by arabinose using kinetic analysis and computational simulation. We hypothesized that the aldehyde (CHO) functional group of arabinose participates in blocking catalysis and inducing unfolding by binding to tyrosinase. Previous findings show the importance of aldehyde groups in tyrosinase inhibition [27], [28], [29] in terms of molecular position, number, and specific interactions with the enzyme; these findings further support our hypothesis. Experimentally, arabinose exerted a mixed-type of inhibition on tyrosinase. Kinetic parameters suggested that arabinose binds to the active site, while measurements of intrinsic and ANS-binding fluorescence revealed significant changes in tertiary structure of tyrosinase. To further explore the docking action of arabinose on tyrosinase, we used computational simulations. Our findings point to new applications for arabinose as a tyrosinase inhibitor.

Section snippets

Materials

Tyrosinase (M.W. 128 kDa), l-DOPA, ANS and arabinose were purchased from Sigma–Aldrich.

Tyrosinase assay

A spectrophotometric tyrosinase assay was performed as previously described [30], [31] in 50 mM sodium phosphate buffer (pH 6.8). To begin the assay, a 10-μl sample of enzyme solution was added to 1 ml of reaction mix. Tyrosinase activity (v) was recorded as the change in absorbance per min at 492 nm using a Perkin Elmer Lambda Bio U/V spectrophotometer.

Kinetic analysis for the mixed-type inhibition

To describe the mixed-type inhibition mechanism, the

Effect of arabinose on tyrosinase activity: inhibition kinetics

Tyrosinase activity was markedly inhibited by arabinose in a complex manner with an IC₅₀ of 0.1 ± 0.07 mM (n = 3) when arabinose was present both in incubating and substrate solutions (Fig. 1). At low arabinose concentrations (less than 0.2 mM), we consistently observed tyrosinase inhibition by up to 60%. At 3.3 mM arabinose, tyrosinase was completely inhibited. When arabinose was omitted from the assay (causing a dilution effect), the tyrosinase activity remained at 20% even with 3.3 mM arabinose in

Discussion

Previous studies recognized the potent inhibitory effect of sugar-based compounds on tyrosinase activity [21], [22], [23], [24], [25]. In this context, we investigated the potentially inhibitory effect of arabinose, a five-carbon sugar containing an aldehyde group, on tyrosinase. We found that arabinose induced changes in K_m, consistent with competitive inhibition through interaction with substrate (l-DOPA) at the active site. Corresponding changes in V_max indicated binding by arabinose to

Acknowledgements

This study was supported by the Zhejiang Provincial Top Key Discipline of Modern Microbiology and Application. Dr. Guo-Ying Qian was supported by the grant of the National Basic Research Program of China (973 Pre-research Program) (2011CB111513). Dr. Hae Young Chung was supported by National Research Foundation of Korea (NRF) grant funded by the Korea Government (MOST) (No. 20090083538) and thanks Aging Tissue Bank for providing research information. Dr. Jun-Mo Yang was supported by a grant of

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Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose

Abstract

Introduction

Section snippets

Materials

Tyrosinase assay

Kinetic analysis for the mixed-type inhibition

Effect of arabinose on tyrosinase activity: inhibition kinetics

Discussion

Acknowledgements

Insect Biochem. Mol. Biol.

Trends Biochem. Sci.

J. Mol. Biol.

J. Biol. Chem.

Int. J. Biol. Macromol.

J. Biotechnol.

Bioorg. Med. Chem. Lett.

Eur. J. Med. Chem.

Bioorg. Med. Chem.

Pigment Cell Melanoma Res.

Pigment Cell Res.

Cell. Mol. Life Sci.

J. Enzyme Inhib. Med. Chem.

Immunol. Rev.

J. Med. Entomol.

Curr. Med. Chem.

Proc. Natl. Acad. Sci. U.S.A.

Proc. Natl. Acad. Sci. U.S.A.

Biochemistry