Characterization of Coppers in CoZ~oZus Consors Lactase

Takeshi Sakurai

College of Liberal Arts and Science, Kunazawa University, Kanazawa, Ishikawa, Japan

ABSTRACT

Lactase was purified from Coliolus censors and its coppers were spectroscopically characterized. While the absorption spectral feature is considerably similar to that of tree lactase, two CD bands coming from the charge transfer (S-+ Cu’+) are inverted. The EPR spectrum and copper analysis indicated that the enzyme contains one type 1, one type 2, and two type 3 coppers, of which the former two types of coppers are EPR detectable. The type 2 copper-depleted enzyme was prepared and characterized. An EPR detectable form of the type 3 coppers was developed from the type 2 copper-depleted derivative at pH 9.5.

INTRODUCTION

Lactase is a multicopper oxidase which is widely distributed in higher plants and fungi. The most extensive studies concerning lactase are of the enzyme from latex of Rhus vernicifera (lacquer tree), which was first discovered as early as 1883 by Yoshida [l]. The main sources for fungi lactase are the Basidomycetes, Polyporus, Pleurotus, Pholiota, etc., and the Ascomycetes, Neurospora, Polyspora, As- pergillus, Podospora, etc. [2]. However, fungi lactase has not been studied in detail except for the enzyme from Polyporus and Neurospora. In the present communication, we show characterization of the coppers involved in the novel lactase from Coliolus censors in comparison with coppers in lactase from Rhus vernicifera.

MATERIALS AND METHODS

Crude lactase obtained from Toyobo Co. was purified by the column chromatogra- phies on DEAE-cellulose, CM-32 cellulose, and Sephadex G-100. The protein sample was homogeneous and gave a single band in analytical electrophoresis test.

Address reprint requests and correspondence to: Dr. Takeshi Sakurai, College of Liberal Arts and Science, Kanazawa University, Marunouchi l-l, Kanazawa, Ishikawa 920, Japan.

Journal of Inorganic Biochemistry, 41,277-281 (1991) 277

0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/91/$3.50

278 T. Sakurai

Wavelength (nm) FIGURE 1. Absorption and CD spectra of native lactase ( ----) and the type 2 copper-de- pleted derivative (-----) (pK 6.0. 0.1 M phosphate buffer).

The final absorption ratio, A,,, /A,, was 18. Enzyme activities were measured for the oxidation of N-ethyl-N,-hydroxymethyl-m-toluidine. One unit causes the forma- tion of micromole of @none dye per minute in acetate buffer (pH 4.5) at 37°C. The activity was increased from 8 to 24 U/mg by purification. Selecrivc removal of the type 2 copper from native enzyme was performed according to the similar method b> Hanna et al. for Polyporus versicolor lactase f.3;

Visible and UV absorbance measurements were taken with a JASCQ Ubest-SU spectrophotometer. CD spectra were measured on a JASCO J-5OK spectropolarirne- ter. The X-band EPR spectra were recorded on a JEOL JES.-REIS system at 77 K. The copper content was determined by using a Jarreli-.Ash AA-1 afomic absorption spectrometer.

RESULTS AND DISCUSSIOK

The molecular weight, Mr value of the purified lactase was determined to be 58000 by SDS polyacrylamide electrophoresis. While Mr value of the tree laccases is in the range of 1 lO.OOO- 14O,OOO, that of the fungi laccases is in the range 6O,OOO-90,000 121. The main difference in Mr values is due to variations in the darbobydraic content. Al! enzymes. except Podospora lactase ;. which is tclrameric, ar- supposed to consist of a single polypeptide chain of almost 500 amino ,lcid residues.

The absorption snectrum IS shown tn Figure i The blue band 15 observed at 608 nm < 5 = 4700 hi! cm 1 nrd shoulders are at (:a. 750 and 1130 rl:ii i pFI 6.0. 0. i M phosphate butfer). While the blue band at 608 nm shifts T nm 10 the shorter wavelength from that of Rhus uernibfera laccasc (615 nm) j41, II shifts only 2 nm

COLIOL US CONSORS LACCASE 279

: 2.003

i-TCNP

0.25 0.3

Mognelic Field (1)

0.35

FIGURE 2. The X-band EPR spectra of native lactase in pH 6.0 phosphate buffer (A), the type 2 copper-depleted derivative in pH 6.0 phosphate buffer (B), and in pH 9.5 Tris-HCl buffer (C). EPR conditions: frequency 9.2 GHz, microwave power 3 mW, modulation 0.79 mT (100 kHz), amplitude 200, temperature 77 K.

from that of Pofyporus versicolor lactase (610 nm) [5]. The shoulder at about 750 nm is more peculiar, compared with that of tree lactase. Although the shoulder at 330 nm is not distinctive for Polyporus lactase, that of the present Coliolus enzyme is very clear as in the case of the tree enzyme. The absorption ratio,

A28O /A6O8 indicated that the content of aromatic amino acids in Coliolus lactase is higher than that in tree lactase.

The CD spectrum showed bands at 750( - ), 605( +), 510( +), 440( - ), and 360( + ) nm (Fig. 1). The number of the bands in the region between 350 to 800 nm is the same as that of Rhus lactase [6] and other multicopper oxidases, ascorbate oxidase [7, 81 and ceruloplasmin [9, lo]. However, of the three bands associated with the charge transfer, S--+Cu 2+ (750 605, and 510 mn bands), the latter two bands are reversed in their signs from those of Rhus lactase, ascorbate oxidase, and ceruloplasmin. Inversion of the three bands due to the type I Cu has been reported for nitrite reductase [l 11.

The EPR spectrum of the native enzyme is shown in Figure 2a. Apparently, two cu2+ species, the type 1 Cu and type 2 Cu are noticed. Spin Hamiltonian parameters for the type 1 Cu are g,, = 2.19, g I = 2.05, A,, = 9.0 x 1O-3 cm-‘; those for the

type2Cuareg,,=2.25,g1 = 2.05, A ,, = 19.1 x 10m3 cm-‘. These parameters for both types of coppers are very similar to those reported for lactase from fungi, Polyporus vercicolor , Podospora anserina, and Neurospora crassa [2]. The A ,,

280 T. Sakurai

value of the type 1 Cu is much greater than that of Rhus vernicifera lactase (A,, = 4.3 x 10. ’ cm I).

Selective removal of the type-2 copper from Coliolus lactase has been success- fully performed according to the similar method for the Polyporus enzyme 131. The total copper content decreased from 4.0 to 2.8 per protein molecule. The amount of EPR detectable copper decreased from 2.0 to 1 .O (the EPR spectrum is shown III Fig. 2b). Resulting absorption and CD spectra are shown in Figure 1 together with those of the native enzyme. Both spectral features in the visible region were hardly changed by depleting the type 2 copper. In addition. the absorption intensity of the shoulder at 330 nm was not drastically decreased, contrary to the case of Rhus lactase, in which the reduction of the type 3 coppers led to the complete disappear- ance of the 330 nm band ] 12, 131. In the present type 2 copper-depleted lactase, the

type 3 coppers seem to be in the oxidized form. The prominent alteration of the CD spectral feature in the 300-400 nm region will come from that the type 2 copper had originally contributed to this region together with the type 1 coppers in the oxrdized form [lo] and/or the steric structure around the type 3 copper site was significantly modified by deleting the type 2 copper. The x-ray crystallographic study of ascorbatc oxidase indicated that the type 2 and type 3 coppers form a trinuclear cluster [I-i/. The presence of the type 2 copper profoundly affects the property and function of the type 3 coppers, the binding and reduction of a molecular oxygen

Recently we studied the effect of pH for native Rhus lactase and Cucumls ascorbate oxidase in detail, having shown that the autoreduction of the type 1 copper occurs gradually at alkaline pH’s 1151. Further. in the case of the type 2 coppcr-de- pleted lactase, a portion of the type 3 coppers became EPR detectable in harmony with the autoreduction of the type 1 copper at high alkaline pH’x 1161. The present coliolus type 2 copper-depicted lactase also gave the type 3 copper EPR signal with the highly rhombic character (g, =: 2.2’7, A, =: 13~3 x !U ’ cm ! I after seven days

incubation at pH 9.5 (Fig. 2~). The spectrum is very similar to that of half met hemocyanin (g, = 2.30, A; = 13.2 x 10 -’ cm ‘) [ 171 and that detected as a reac- tion intermediate of the type 2 copper-depleted lactase (g, = 2.28. A I : 13.0 x IO ’ cm ‘1 [18].

The author expresses his sincere thanks to Tqvoho Co. for furnishing Coliolus laccase.

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Received August 6, 1990. accepted August 14, 1990