ANALYTICAL
BIOCHEMISTRY
194,
41-44
(19%)
Photochemical cis-trans lsomerization of Furylacryloylpeptides and Their Different Kinetic Behavior as Substrates for Carboxypeptidase Y Anders
Kanstrup
and Ole Buchardtl
Research Center for Medical Biotechnology, Chemical Laboratory Universitetsparken 5, DK-2100 0, Copenhagen, Denmark
Received
October
22, 1990
Furylacryloyl substrates used ments of proteolytic enzymes are photoisomerize quickly in plain matic transformation of the two different to cause problems for without careful protection against Press.
II, The H. C. 0r.sted Institute,
in kinetic measureshown to cis-trans daylight. The enzyforms is sufficiently such measurements light. o 1991 Academic
Inc.
INTRODUCTION
During the kinetic analysis of a series of chemically modified derivatives of carboxypeptidase Y (CPD-Y)2 (EC 3.4.16.1), we were unable to reproduce the K, and Jz~~for the native enzyme cited in the literature (1) and obtained unacceptably large interexperimental variations. This enzyme catalyzes the liberation of the carboxy-terminal amino acid from peptides, with a preference for substrates with large hydrophobic residues, and we therefore used 3-(2-furylacryloyl)-~-phenylalanylL-leucine (FA-Phe-Leu) as our standard substrate. The use of substrates, in which the cromogenic reporter group furylacrylic acid (FA) is connected via an amido bond to the N-terminal of a dipeptide or an amino acid ester, was originally proposed by Bernhard et al. (2) and McClure and Neurath (3) and has since gained a widespread use in enzyme kinetics (4). Many such substances have been reported in the literature (5) and several of these are commercially available. However, a solution prepared for enzyme kinetics of FA-Phe-Leu
1 To whom correspondence should be addressed. ’ Abbreviations used: CPD-Y, carboxypeptidase methyl sulfoxide; EDTA, ethylenediaminetetraacetic lacrylic acid; Mes, 2-(IV-morpholino)ethanesulfonic 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Y; DMSO, diacid, FA, furyacid.
placed at the workbench in daylight suffered a 32% decrease in A,,, during an hour, and we were thus prompted to investigate the influence of this light sensitivity upon the kinetic characterizations of the enzymes. The photochemistry of 3-(2-furyl)acrylic acid has been briefly investigated previously, where it was found that trans-furylacrylic acid in solution cisltrans photoisomerizes (6,7) and in the solid state photodimerizes (E&9). Thus, light-induced transformations of the furylacrylic acid-based substrates may be a source of error in enzyme kinetics.
MATERIALS
AND
METHODS
FA-Phe-Leu and FA-Gly were prepared as in (10). CPD-Y was a gift from Carlbiotech A/S, Copenhagen, which is gratefully acknowledged. ‘H NMR spectra were recorded on a 250-MHz Bruker instrument. Ultraviolet spectra and enzyme kinetics were recorded on a PerkinElmer X-17 spectrometer at 25°C. Enzyme kinetics were performed in 50 mM Mes, 1 ITIM EDTA, 0.1% DMSO, pH 6.5. Unless otherwise specified, substrate and buffer solutions were prepared in the dark, kept in brown bottles, and mixed directly in the cuvette just before use. K,,, and k,, were extracted from initial rate data using the Enzfitter data analysis program from Elsevier Biosoft. Small-scale photolysis was performed on standard samples (see below) using 325-nm light from a Photon Technology International 200 W light source, equipped with a monochromator, until a steady state was reached. Typically, this took ~30 min with the low-intensity monochromatic light employed. Preparative photolysis was performed until steady state using a Rayonet RPR208 preparative photoreactor equipped with 300-nm fluorescent tubes. Daylight photolysis was performed with a 0.04 mM sample in a quartz cuvette covered with a 41
Inc. reserved.
42
KANSTRUP
AND TABLE
Spectroscopic,
Photochemical,
and Kinetic
Properties
‘H NMR 6: G-M 6: (HA J: (-CH=CH-) uv LX i&0.1 mM) Photochemistry k (pM/min); 300 nm (isosbestic k (rM/min); daylight Enzyme kinetics K, bM) k,, (min-‘) AA(332 nm)/mM
point)
1
of trans-FA-Phe-Leu,
trans-FA-Gly,
and ck-FA-Gly
ck-FA-Phe-Leu
7.23 ppm (d, 1H) 6.54 ppm (d, 1H)
6.63 ppm (d, 1H) 5.92 ppm (d, 1H)
7.34 ppm (d, 1H) 6.57 ppm (d, 1H)
15.6 Hz
12.9 Hz
15.6 Hz
5.97 ppm 12.9 Hz
303 nm 23,300 2.296
282 nm 10,300 0.928
70 X 10m3 (274 nm)
30 x lo+
54 X 10e3 (275 nm)
29 x lo-+
13 x 10-3
4.9 x 10-3
300 nm and thus give little protection for compounds which, as in this case, absorb above this wavelength. Enzymology. Utilizing preparative HPLC, it was possible to isolate enough of the pure cis-FA-Phe-Leu from the product mixture to perform a kinetic characterization toward our standard enzyme CPD-Y, the results of which are summarized in Table 1. At 332 nm, where the largest hA/mM upon hydrolysis occurs for the trans form, the corresponding cis change is 3.7 times smaller. Furthermore, k,,(cis) = 2400/min and K,,,(cis) = 0.024 mM, whereas k,,,(trans) = 5500/min and K,(trans) < 0.010 InM, the reason for the inaccuracy being that the AA,,, for the total hydrolysis of a 0.010 mM solution of trans-FA-Phe-Leu is 0.010 OD, which prevents sufficient measurements of more dilute solutions. For two substrates competing for the same active site the relative rates of hydrolysis will depend on their relative k=,,,/K, (12). Thus, in the case of CPD-Y, even in a
h. 1
0 0
FIG. 4.
IS1
Calculated V/[S] plot for CPD-Y toward trans-FA-PheLeu. Without photolysis (A). All substrate solutions 50% photolyzed, e.g., from the making of a stock solution (6). A 10% increase ofphotolysis for each solution until steady state (w). Arbitrary units.
44
KANSTRUP
AND
1:l cisltruns mixture the tram form rate of hydrolysis will be five times faster than the cis rate. This, combined with the truns form having a much larger AA,,,lmmol upon hydrolysis, leads to initial rates observed from even strongly photolyzed samples which may well be correct in the sense that they are due to the hydrolysis of the trans form only, but they will originate from much lower substrate concentrations. However, no general predictions can be made, since the actual influence of this phenomenon will depend on the individual conditions, enzymes, etc. (Fig. 4). With other enzymes the cis form may have the largest k,,IK,. In such cases errors might become extreme if a AA/mm01 conversion factor, determined from freshly made solutions of the pure trams form, is used with substrate dilutions which have only been kept on ice and not protected from light before use. On the basis of these findings, we recommend that stock solutions be prepared in the dark and kept in brown bottles, and that appropriate dilutions be prepared directly into the cuvette under low-light conditions. It is obvious that due care must be taken whenever these types of substrates are used. However, an examination of the photolability under the actual working conditions is very easily performed by uv spectroscopy and is highly recommended.
BUCHARDT
ACKNOWLEDGMENTS This work was supported to A. Kanstrup. We thank cussions.
by a grant Dr. Klaus
from the Carlsberg Breddam for many
Foundation helpful dis-
REFERENCES 1. Breddam, 2. Bernhard, 1108-1118. 3. McClure, 4. Pozdnev, ences cited 5. CAS-Online
K. (1984) S. A., Lau,
Curlsberg
W., and Neurath, V. F. (1986) therein.
Res. Commun.
S. J., and Noller, H. (1966)
Zh. Obshch.
4,535-54.
H. (1965)
Biochemistry
Biochemistry
Khim.
4,
5,1425-1438.
56,690-695,
and refer-
search (1990) 195 answers. 6. Karminski-Zamola, G., and Jakopcic, K. (1974) Croat. Chem. Actu 46, 71-78. 7. Norval, M., Simpson, T. J., Bardshiri, E., and Howie, S. E. M. (1989) Photochem. Photobid. 49,633-639. 8. Ghosh, U., and Misra, T. N. (1988) J. Polymer Sci. Part A. 26, 1681. 9. Lahav, M., and Schmidt, G. M. J. (1969) J. Chem. Sot. B. 239-43. 10. Blumberg, S., and Valle, B. L. (1975) Biochemistry 14, 2419. 11. Atkins, P. W. (1982) Physical Chemistry, p. 938, Oxford Press, London/New York. 12. Fersht, Freeman,
A. (1985) Enzyme San Francisco.
Structure
and
Mechanism,
2410Univ. p. 112,
BIOCHEMISTRY
194,
41-44
(19%)
Photochemical cis-trans lsomerization of Furylacryloylpeptides and Their Different Kinetic Behavior as Substrates for Carboxypeptidase Y Anders
Kanstrup
and Ole Buchardtl
Research Center for Medical Biotechnology, Chemical Laboratory Universitetsparken 5, DK-2100 0, Copenhagen, Denmark
Received
October
22, 1990
Furylacryloyl substrates used ments of proteolytic enzymes are photoisomerize quickly in plain matic transformation of the two different to cause problems for without careful protection against Press.
II, The H. C. 0r.sted Institute,
in kinetic measureshown to cis-trans daylight. The enzyforms is sufficiently such measurements light. o 1991 Academic
Inc.
INTRODUCTION
During the kinetic analysis of a series of chemically modified derivatives of carboxypeptidase Y (CPD-Y)2 (EC 3.4.16.1), we were unable to reproduce the K, and Jz~~for the native enzyme cited in the literature (1) and obtained unacceptably large interexperimental variations. This enzyme catalyzes the liberation of the carboxy-terminal amino acid from peptides, with a preference for substrates with large hydrophobic residues, and we therefore used 3-(2-furylacryloyl)-~-phenylalanylL-leucine (FA-Phe-Leu) as our standard substrate. The use of substrates, in which the cromogenic reporter group furylacrylic acid (FA) is connected via an amido bond to the N-terminal of a dipeptide or an amino acid ester, was originally proposed by Bernhard et al. (2) and McClure and Neurath (3) and has since gained a widespread use in enzyme kinetics (4). Many such substances have been reported in the literature (5) and several of these are commercially available. However, a solution prepared for enzyme kinetics of FA-Phe-Leu
1 To whom correspondence should be addressed. ’ Abbreviations used: CPD-Y, carboxypeptidase methyl sulfoxide; EDTA, ethylenediaminetetraacetic lacrylic acid; Mes, 2-(IV-morpholino)ethanesulfonic 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Y; DMSO, diacid, FA, furyacid.
placed at the workbench in daylight suffered a 32% decrease in A,,, during an hour, and we were thus prompted to investigate the influence of this light sensitivity upon the kinetic characterizations of the enzymes. The photochemistry of 3-(2-furyl)acrylic acid has been briefly investigated previously, where it was found that trans-furylacrylic acid in solution cisltrans photoisomerizes (6,7) and in the solid state photodimerizes (E&9). Thus, light-induced transformations of the furylacrylic acid-based substrates may be a source of error in enzyme kinetics.
MATERIALS
AND
METHODS
FA-Phe-Leu and FA-Gly were prepared as in (10). CPD-Y was a gift from Carlbiotech A/S, Copenhagen, which is gratefully acknowledged. ‘H NMR spectra were recorded on a 250-MHz Bruker instrument. Ultraviolet spectra and enzyme kinetics were recorded on a PerkinElmer X-17 spectrometer at 25°C. Enzyme kinetics were performed in 50 mM Mes, 1 ITIM EDTA, 0.1% DMSO, pH 6.5. Unless otherwise specified, substrate and buffer solutions were prepared in the dark, kept in brown bottles, and mixed directly in the cuvette just before use. K,,, and k,, were extracted from initial rate data using the Enzfitter data analysis program from Elsevier Biosoft. Small-scale photolysis was performed on standard samples (see below) using 325-nm light from a Photon Technology International 200 W light source, equipped with a monochromator, until a steady state was reached. Typically, this took ~30 min with the low-intensity monochromatic light employed. Preparative photolysis was performed until steady state using a Rayonet RPR208 preparative photoreactor equipped with 300-nm fluorescent tubes. Daylight photolysis was performed with a 0.04 mM sample in a quartz cuvette covered with a 41
Inc. reserved.
42
KANSTRUP
AND TABLE
Spectroscopic,
Photochemical,
and Kinetic
Properties
‘H NMR 6: G-M 6: (HA J: (-CH=CH-) uv LX i&0.1 mM) Photochemistry k (pM/min); 300 nm (isosbestic k (rM/min); daylight Enzyme kinetics K, bM) k,, (min-‘) AA(332 nm)/mM
point)
1
of trans-FA-Phe-Leu,
trans-FA-Gly,
and ck-FA-Gly
ck-FA-Phe-Leu
7.23 ppm (d, 1H) 6.54 ppm (d, 1H)
6.63 ppm (d, 1H) 5.92 ppm (d, 1H)
7.34 ppm (d, 1H) 6.57 ppm (d, 1H)
15.6 Hz
12.9 Hz
15.6 Hz
5.97 ppm 12.9 Hz
303 nm 23,300 2.296
282 nm 10,300 0.928
70 X 10m3 (274 nm)
30 x lo+
54 X 10e3 (275 nm)
29 x lo-+
13 x 10-3
4.9 x 10-3
300 nm and thus give little protection for compounds which, as in this case, absorb above this wavelength. Enzymology. Utilizing preparative HPLC, it was possible to isolate enough of the pure cis-FA-Phe-Leu from the product mixture to perform a kinetic characterization toward our standard enzyme CPD-Y, the results of which are summarized in Table 1. At 332 nm, where the largest hA/mM upon hydrolysis occurs for the trans form, the corresponding cis change is 3.7 times smaller. Furthermore, k,,(cis) = 2400/min and K,,,(cis) = 0.024 mM, whereas k,,,(trans) = 5500/min and K,(trans) < 0.010 InM, the reason for the inaccuracy being that the AA,,, for the total hydrolysis of a 0.010 mM solution of trans-FA-Phe-Leu is 0.010 OD, which prevents sufficient measurements of more dilute solutions. For two substrates competing for the same active site the relative rates of hydrolysis will depend on their relative k=,,,/K, (12). Thus, in the case of CPD-Y, even in a
h. 1
0 0
FIG. 4.
IS1
Calculated V/[S] plot for CPD-Y toward trans-FA-PheLeu. Without photolysis (A). All substrate solutions 50% photolyzed, e.g., from the making of a stock solution (6). A 10% increase ofphotolysis for each solution until steady state (w). Arbitrary units.
44
KANSTRUP
AND
1:l cisltruns mixture the tram form rate of hydrolysis will be five times faster than the cis rate. This, combined with the truns form having a much larger AA,,,lmmol upon hydrolysis, leads to initial rates observed from even strongly photolyzed samples which may well be correct in the sense that they are due to the hydrolysis of the trans form only, but they will originate from much lower substrate concentrations. However, no general predictions can be made, since the actual influence of this phenomenon will depend on the individual conditions, enzymes, etc. (Fig. 4). With other enzymes the cis form may have the largest k,,IK,. In such cases errors might become extreme if a AA/mm01 conversion factor, determined from freshly made solutions of the pure trams form, is used with substrate dilutions which have only been kept on ice and not protected from light before use. On the basis of these findings, we recommend that stock solutions be prepared in the dark and kept in brown bottles, and that appropriate dilutions be prepared directly into the cuvette under low-light conditions. It is obvious that due care must be taken whenever these types of substrates are used. However, an examination of the photolability under the actual working conditions is very easily performed by uv spectroscopy and is highly recommended.
BUCHARDT
ACKNOWLEDGMENTS This work was supported to A. Kanstrup. We thank cussions.
by a grant Dr. Klaus
from the Carlsberg Breddam for many
Foundation helpful dis-
REFERENCES 1. Breddam, 2. Bernhard, 1108-1118. 3. McClure, 4. Pozdnev, ences cited 5. CAS-Online
K. (1984) S. A., Lau,
Curlsberg
W., and Neurath, V. F. (1986) therein.
Res. Commun.
S. J., and Noller, H. (1966)
Zh. Obshch.
4,535-54.
H. (1965)
Biochemistry
Biochemistry
Khim.
4,
5,1425-1438.
56,690-695,
and refer-
search (1990) 195 answers. 6. Karminski-Zamola, G., and Jakopcic, K. (1974) Croat. Chem. Actu 46, 71-78. 7. Norval, M., Simpson, T. J., Bardshiri, E., and Howie, S. E. M. (1989) Photochem. Photobid. 49,633-639. 8. Ghosh, U., and Misra, T. N. (1988) J. Polymer Sci. Part A. 26, 1681. 9. Lahav, M., and Schmidt, G. M. J. (1969) J. Chem. Sot. B. 239-43. 10. Blumberg, S., and Valle, B. L. (1975) Biochemistry 14, 2419. 11. Atkins, P. W. (1982) Physical Chemistry, p. 938, Oxford Press, London/New York. 12. Fersht, Freeman,
A. (1985) Enzyme San Francisco.
Structure
and
Mechanism,
2410Univ. p. 112,
