Pressure and temperature-induced denaturation of carboxypeptidase Y and procarboxypeptidase Y

Trends in High PressureBioscienceand Biotechnology R. Hayashi(editor) 9 2002 ElsevierScienceB.V. All rights reserved.

33

Pressure and t e m p e r a t u r e - i n d u c e d denaturation o f carboxypeptidase Y and procarboxypeptidase Y Michiko Kato, Rikimaru Hayashi, Reinhard Lange*, and Claude Balny* Laboratory of Biomacromolecules, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan INSERM U-128, IFR 24, 1919 Route de Mende (CNRS), F-34293 Montpellier, Cedex 5, France

Pressure-induced denaturation of carboxypeptidase Y (CPY) and procarboxypeptidase Y (proCPY) was investigated by measuring the intrinsic fluorescence in the temperature range of 20-80~ or the pressure range of 0.1-700 MPa.

The pressure-induced denaturation

of CPY showed at least a three-state transition, while temperature-induced denaturation of CPY showed a two-state transition.

The pressure and temperature-induced denaturation of

proCPY showed a simple two-state transition.

1. INTRODUCTION Carboxypeptidase Y (CPY), a vacuolar enzyme from Saccharomyces cerevisiae belongs to the family of serine proteases (1).

Procarboxypeptidase Y (proCPY) is a

precursor of CPY which consists of a mature region (421 amino acid residues) and a propeptide of 91 amino acid residues at the N-terminus.

The propeptide is an essential for

folding, as has been shown by both in vivo and in vitro studies (2-4), and for maintaining CPY in an inactive state. The intrinsic protein fluorescence which is mainly due to tryptophan and tyrosine residues (5) reflects the conformation of proteins: the wavelength of the maximum fluorescence, the center of the spectral mass, changes depending on the polarity of the

34 environment of these residues (6).

Since CPY contains 10 tryptophan and 24 tyrosine

residues, which are evenly distributed throughout the entire protein molecule, the denaturation of CPY can be measured by changes in its intrinsic fluorescence (7). In

this

report,

differences

between

pressure-induced

denaturation

and

temperature-induced denaturation of CPY and proCPY were observed by measuring fluorescence changes and the conformation of the two proteins is discussed.

2. EXPERIMENTAL PROCEDURES

Materials: Carboxypeptidase Y (CPY) was obtained from Oriental Yeast Co. (Lot 21003805) (Osaka, Japan).

ProCPY was purified from yeast strain BJ2168 transformed by

pTSY3 in our laboratory.

3-(N-Morpholino) propanesulphonic acid, Mops, was obtained

from Nacalai Tesque (Kyoto, Japan). Fluorescence spectroscopy: Fluorescence measurements of CPY and proCPY were made using an Aminco-Bowmann Series 2 luminescence spectrometer (SLM), which was modified to accommodate a pressure cell.

Protein concentrations of 0.1mg/ml in 50 mM

Mops buffer (pH 7.0) were used for the pressure and temperature measurements, and measurements were made using a 5 mm diameter quartz cuvette. temperature was increased, typically in steps of about 50 MPa or 5 ~

The pressure or respectively and the

fluorescence of the protein was observed after a 5 min pause to allow conformational equilibrium to be attained.

Intrinsic fluorescence was measured by exciting at 280 nm (4

nm slit) and emission was recorded between 310-410 nm (4 nm slit, 1 nm step size).

The

fluorescence spectra of the sum of the tryptophan and tyrosine contributions were quantified by specifying the center of the spectral mass, , as defined and used by Weber and coworkers as follows (8): =s

F~/EF~

where vi is the wavenumber and F~ is the fluorescence intensity at ui. Tm and Pm are temperature and pressure, respectively, which is a half of the center of the spectral mass.

35 Table 1 Effects of pressure and temperature on CPY and proCPY In situ experiments

CPY Pm (MPa) determined by fluorescence __A_ct!_~!t__y___(__~

340 b

proCPY

by fluorescence

Tm (~

by CD at 222 nm b

CPY

proCPY

253

p_ress_ure tr_e at_me_nt_~ ..........................................................

Tm (~

Ex situ experiments

58.6

54.5

57

57

9O_.8_ ............. 4_8_._1_.........

Activity (%) after heat treatment c a Activity after treatment at 400 MPa and 25 ~ for 10 min (10). bTaken from reference (9). c Activity after treatment at 50 ~ for 1 hr (10).

35.8

35.6

3. RESULTS

3.1. In situ temperature-induced changes of CPY and proCPY The intrinsic fluorescence spectra of CPY and proCPY were measured against temperature up to 80 ~ at 0.1 MPa.

The unfolding curves of CPY and proCPY showed a

simple two-state transition with a Tm of 58.6 and 54.5 ~

respectively

(Table 1).

The Tm

of proCPY was found to be about 4 ~ lower than that of CPY, indicating that proCPY was less stable than CPY to temperature.

By decreasing the temperature from the highest

temperature tested, changes in <~> of CPY and proCPY were partially reversible. Based on measurement of the circular dichroism (CD) at 222 nm with an increase in temperature at a rate of 0.5 ~

CPY and proCPY also showed the same Tm at 57 ~ (9).

3.2. In situ pressure-induced changes of CPY and proCPY The intrinsic fluorescence spectra of CPY and proCPY were measured against pressure up to 700 MPa at 25 ~

The <~> of CPY was red-shifted with increasing pressure,

indicating exposure of the tryptophan and tyrosine residues to the solvent.

The unfolding of

CPY showed at least a three-state transition, and did not follow a simple two-state transition. The complete unfolding of CPY was not observed at pressures up to 700 MPa at 25 ~ Based on the clear sigmoidal shape of the second transition, the Pm2 was calculated as to be

36 340 MPa (Table 1).

The Pm of the first transition, Pml, was estimated at low pressures of

below 50 MPa and the Pm of the third transition, Pm3, was observed at 540 MPa or higher. The <~> of proCPY was red-shitted with an increase in pressure and showed a simple two-state transition with a Pm of 253 MPa (Table 1).

After the release of pressure from the

highest pressure tested, changes in of CPY and proCPY were incompletely reversible.

3.3. Ex situ temperature-and pressure-induced changes of CPY and proCPY After temperature treatment at 45 ~

for lhr, the activities of CPY and proCPY

remained at 90.6% and 83.2%, respectively (data not shown).

After temperature treatment

at 50 ~ for lhr, activities of CPY and proCPY were determined to be 35.8% and 35.6%, respectively (Table 1)(10).

These data indicate that there are no significant differences

between CPY and proCPY with respect to temperature treatment. After pressure treatment at 400 MPa and 25 ~

for 10 min, the activity of CPY

remained at 90.8%, but that of proCPY decreased to 48.1%, activity of which was measured after activation using proteinase K (10).

These data indicate that CPY is more stable than

proCPY with respect to pressure treatment.

4. DISCUSSION

The temperature-induced denaturation curves of CPY and proCPY showed a simple two-state transition with similar Tm values.

These results indicate that temperature induces

the cooperative denaturation of proteins. The pressure-induced denaturation of CPY showed at least a three-state transition with increases in pressure up to 700 MPa.

Relatively low pressures (below 150 MPa) induced a

small change in <~>, indicating only small conformational changes in CPY.

This finding is

consistent with a previous report (7) in which it was shown that no hydrophobic core was exposed, as evidenced by no increase in ANS-binding fluorescence and by only a small loss in catalytic activity,

Higher pressure, 150 to 500 MPa, induced a large decrease in ,

indicating a large conformational change.

At these pressures, it has been demonstrated that

ANS-binding fluorescence increased with a decreased activity (7).

This result supports the

view that the pressure-denatured CPY molecule at 150-500 MPa exists in a molten globule-like state (11).

A multiple transition via the molten-globule has also been reported

37 for other proteins (12, 13). Higher pressures in excess of 500 MPa induced a larger red-shift in , although the protein appeared to be incompletely denatured.

The three-state

transition seemingly reflects a sequential unfolding of at least two structural domains (7). That is, two domains, one of which is the 13-sheet-rich central core and another of which is the helix-rich external part of CPY molecule, unfold independently. On the other hand, the pressure-induced denaturation of proCPY showed a simple two-state transition.

The difference between the pressure denaturation of CPY and proCPY

may be explained as follows.

The propeptide sterically covers the active site, and thereby

prevents substrate binding, as is the case for aspartic proteinase (14) and subtilisin (14, 15). Although the X-ray crystal structure of proCPY has not yet been solved, the charge-relay system of the mature CPY seems to be incompletely formed by some interference of the propeptide (16).

This suggests that the cleft of the active site, which is located at the

interface of the two domains is covered with the propeptide and, as a result, proCPY behaves as a one domain, resulting in cooperative unfolding. In conclusion, CPY is composed of two structural domains, which show different sensitivities to pressure.

Therefore pressure induces a multi step conformational change.

On the other hand, proCPY denatures via a two-state transition as the propeptide combines the two structural domains so as to form one domain.

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