Proc. Natl. Acad. Sci. USA
Vol. 74, No. 8, pp. 3273-3277, August. 1977 Biochemistry
Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: Determination of the nature of the azotyrosyl-248*zinc complex (active site/conformation/intramolecular coordination)
R. K. SCHEULE*, H. E. VAN WARTt, B. L. VALLEEt, AND H. A. SCHERAGA* * Department of Chemistry, Cornell University, Ithaca, New York 14853; and t Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115
Contributed by H. A. Scheraga, May 26, 1977
Resonance Raman spectra of arsanilazotyroABSTRACT syl-248 carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) exhibit only the vibrational bands of its chromophoric azotyrosyl-248 residue uncomplicated by background interference from either water or other components of the protein. The resonance Raman spectra contain multiple, discrete bands which change as a function of pH, thereby demonstrating the existence of interconvertible species of the azotyrosine probe in solution. S ectra of model azophenols and of the apoazoenzyme establish the identity of these species. All conclusions about the azoenzyme based on the resonance Raman spectra, including the apparent pK values for the interconversion of these species, are in complete agreement with those drawn earlier from studies by absorption spectroscopy. In addition, the properties of resonance Raman bands that have been identified with the motions of specific atoms of azotyrosyl-248 provide details of the interactions of specific atoms of this chromophore with the catalytic zinc atom at the active site. In particular, this has allowed elucidation of the structure of the azotyrosyl-248-zinc coordination complex. Such experiments are also providing information on the effects of crystallization on the enzyme and on its interaction with inhibitors. The important potential of resonance Raman spectroscopy for the study of the structure of chromophoric components of active enzymatic sites and of metal complex ions is discussed.
Chemical modification of carboxypeptidase A (peptidyl-Lamino-acid hydrolase, EC 3.4.12.2) with diazotized p-arsanilic acid specifically labels the tyrosine residue at position 248, with retention of catalytic activity (1). The resulting arsanilazotyrosyl-248 carboxypeptidaset exhibits characteristic visible absorption and circular dichroic spectra (2) whose pH dependence has permitted identification of several species of the azotyrosine probe in solution (3). In particular, the azotyrosyl-248 residue and the active-site zinc atom form a spectrally distinct, intramolecular coordination complex whose stability is maximal at pH 8.5. Substrates, inhibitors, crystallization, variations in pH, and denaturation disrupt this complex and produce spectral changes (3-5), thereby making azotyrosyl-248 a probe of the active site. We have now used resonance Raman (rR) spectroscopy to study in detail the microenvironment of azotyrosyl-248 and the structure of its zinc complex. This chromophoric, enzymatically active enzyme is ideally suited for such studies because its absorption and circular dichroic spectra are well known (3-5) and provide a firm basis for the interpretation of the rR spectra. Excitation with wavelengths that are in resonance with the visible electronic transitions of the azotyrosyl-248 chromophore selectively and markedly enhances the intensities of those The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Raman-active vibrational modes that are coupled to the electronic transitions. The resultant rR spectra of this enzyme reinforce conclusions based on the absorption and circular dichroic spectra obtained earlier. In addition, the behavior of vibrational bands that have now been assigned to and identified with the motions of specific atoms of the chromophoric ligand provide new information about the chemical details of the interaction of azotyrosyl-248 with the catalytic zinc atom of the active site. Work on the crystalline azoenzyme, on the interaction of the azoenzyme with inhibitors, and on model azophenols has also been completed and bands have been assigned (R. K. Scheule, H. E. Van Wart, B. 0. Zweifel, B. L. Vallee, and H. A. Scheraga, unpublished data). MATERIALS AND METHODS Carboxypeptidase A, prepared by the method of Cox et al. (6), was obtained as a crystal suspension (Sigma Chemical Co.) and purified by affinity chromatography (7). Its arsanilazo derivative was synthesized by treating a suspension of the native enzyme crystals with diazotized p-arsanilic acid as described (8). Apoarsanilazocarboxypeptidase was prepared by dialysis against 1,10-phenanthroline (9). Protein concentration was measured by the absorbance at 278 nm with E278 = 7.32 X 104 M-' cm-' for both the zinc azoenzyme and the apoazoenyme (4). Monotetraazolylazo-N-acetyltyrosine (TAT) was synthesized as described (10), and its concentration was determined with f416 = 4.39 X 103 M- cm-' (3). All rR experiments were performed under metal-free conditions. Buffers were extracted with 0. 1% Dithizon in carbon tetrachloride to remove traces of metals. Quartz sample tubes were soaked in concentrated nitric and sulfuric acids (1:1) and rinsed with metal-free water (11). The concentration of the azochromophore was about 0.06 mM in 2 mM Tris-HCI/0.5 M NaCl and 0.05 M in Na2SO4, the latter serving as an internal frequency and intensity standard. The rR spectra were obtained by using excitation from the 488.0 nm line of a Coherent Radiation argon ion laser. The power at the sample was 10-40 mW. To minimize the absorption of the incident and scattered radiations, the sample was oriented obliquely to the incident laser beam. The light scattered from the surface of the sample tube at 900 to the incident beam was focused on the entrance slit of a Spex 1401 double monochromator operated in a stepping mode. The monoAbbreviations: rR, resonance Raman; TAT, monotetraazolylazo-Nacetyltyrosine. t "Arsanilazotyrosyl-248 carboxypeptidase," "arsanilazocarboxy-
peptidase," "zinc azoenzyme," and "azoenzyme" all are terms used
interchangeably for monoarsanilazotyrosyl-248 zinc carboxypeptidase; "apoazoenzyme" is used interchangeably for apoarsanilazotyrosyl-248 carboxypeptidase. 3273
3274
Proc. Nati. Acad. Sci. USA 74 (1977)
Biochemistry: Scheule et al.
chromator and photomultiplier tube were interfaced to an Interdata 70 computer. Typically, photons were counted at each frequency for 1 min and the data were stored in digital form on magnetic tape cassettes. Spectra were plotted from these data by using programs with noise-smoothing and scale-varying capabilities. Resolution was 8 cm-1 in all cases. All band positions and molar intensities were determined relative to the 981 cm-1 symmetric stretching mode of the sulfate ion (12). Sharp bands have an accuracy of ±1 cm-1. All spectra were obtained at 200C, except that those of the apoazoenzyme were recorded at 5°C to prevent denaturation in the course of the rR experiment. Spectra obtained from consecutive scans of any given sample were reproducible, and photodecomposition was not observed. The peptidase and esterase activities of the arsanilazoenzyme were identical before and after exposure to the laser beam.
c
0 0
450
Wavelength (nm)
RESULTS AND DISCUSSION rR Characterization of Azotyrosyl-248 Species. The existence of three interconvertible species of the azotyrosyl-248 residue of the zinc azoenzyme has been demonstrated by means of absorption and circular dichroism spectroscopy (3, 5). Titration of the zinc azoenzyme from pH 6 to 11 converts the protonated azophenol first to the intramolecular azophenolate-zinc complex and, ultimately, to the free azophenolate ion (Fig. 1 upper). The vibrational bands in the 1000-1600 cm-' region of the rR spectra of the azoenzyme distinguish and characterize these three species over this same pH range (Fig. 1 lower). At intermediate pH values the rR spectra were superimpositions of these three basic spectra. Many of the rR bands of all three species are virtually the same; however, several differ characteristically and so are diagnostically valuable. These differences consist of the presence or absence of certain bands and changes in frequency or intensity, or both, of others. We shall first describe the rR characteristics of the three azotyrosyl-248 species, based on band assignments deduced from the Raman, rR, and infrared spectra of several model azophenols and their 15N and 2H derivatives. Zinc Arsanilazotyrosyl-248 Carboxypeptidase A. All three species of azotyrosyl-248 exhibit a very strong band at about 1430 cm-', due to an N=-N stretching motion. The exact frequency of this band is characteristic of the particular species present; the coexistence of more than one species of the azoprobe can be inferred from the existence of multiple bands in this region. All three species also display a band due to a C-N stretching motion at about 1389 cm-'; formation of the complex greatly enhances its intensity. In addition, all the rR spectra exhibit a doublet with bands near 1150 and 1160 cm-1 whose intensity ratio is a useful index of the species present, even though the individual frequencies remain largely independent of pH. The formation of the azotyrosyl-248-zinc complex coincides with the appearance of three sharp rR bands that are either nonexistent or very weak in the spectra of the other species. The most intense of these occurs at 1338 cm-'. It is absent at low pH and is broad and poorly defined at high pH. The weak band at 13M5 cm-' (Fig. 1 lower) is due to the small amount of the zinc complex that is present at pH 6.2.§ Another as yet unassigned band, associated only with the complex, occurs at 1212 cm-1, § The relative resonance enhancements of the three azoprobe species were found to be proportional to their molar absorptivities at 488.0 nm (see Fig. 1 upper). Hence, the small amount of the zinc azoenzyme complex present at pH 6.2 contributes measurably to the total
rR scattering observed.
pH 10.8
pH 8.5
1
3
V
386~~~~~~o I
1538
~~~~~~1216
Il LI
~~~~~~~~~~1150 1120
1486
1314
0y 7
12
pH 6.2 o456
'339~~~~~~13 ' iJ
1500
Vol. 74, No. 8, pp. 3273-3277, August. 1977 Biochemistry
Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: Determination of the nature of the azotyrosyl-248*zinc complex (active site/conformation/intramolecular coordination)
R. K. SCHEULE*, H. E. VAN WARTt, B. L. VALLEEt, AND H. A. SCHERAGA* * Department of Chemistry, Cornell University, Ithaca, New York 14853; and t Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115
Contributed by H. A. Scheraga, May 26, 1977
Resonance Raman spectra of arsanilazotyroABSTRACT syl-248 carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) exhibit only the vibrational bands of its chromophoric azotyrosyl-248 residue uncomplicated by background interference from either water or other components of the protein. The resonance Raman spectra contain multiple, discrete bands which change as a function of pH, thereby demonstrating the existence of interconvertible species of the azotyrosine probe in solution. S ectra of model azophenols and of the apoazoenzyme establish the identity of these species. All conclusions about the azoenzyme based on the resonance Raman spectra, including the apparent pK values for the interconversion of these species, are in complete agreement with those drawn earlier from studies by absorption spectroscopy. In addition, the properties of resonance Raman bands that have been identified with the motions of specific atoms of azotyrosyl-248 provide details of the interactions of specific atoms of this chromophore with the catalytic zinc atom at the active site. In particular, this has allowed elucidation of the structure of the azotyrosyl-248-zinc coordination complex. Such experiments are also providing information on the effects of crystallization on the enzyme and on its interaction with inhibitors. The important potential of resonance Raman spectroscopy for the study of the structure of chromophoric components of active enzymatic sites and of metal complex ions is discussed.
Chemical modification of carboxypeptidase A (peptidyl-Lamino-acid hydrolase, EC 3.4.12.2) with diazotized p-arsanilic acid specifically labels the tyrosine residue at position 248, with retention of catalytic activity (1). The resulting arsanilazotyrosyl-248 carboxypeptidaset exhibits characteristic visible absorption and circular dichroic spectra (2) whose pH dependence has permitted identification of several species of the azotyrosine probe in solution (3). In particular, the azotyrosyl-248 residue and the active-site zinc atom form a spectrally distinct, intramolecular coordination complex whose stability is maximal at pH 8.5. Substrates, inhibitors, crystallization, variations in pH, and denaturation disrupt this complex and produce spectral changes (3-5), thereby making azotyrosyl-248 a probe of the active site. We have now used resonance Raman (rR) spectroscopy to study in detail the microenvironment of azotyrosyl-248 and the structure of its zinc complex. This chromophoric, enzymatically active enzyme is ideally suited for such studies because its absorption and circular dichroic spectra are well known (3-5) and provide a firm basis for the interpretation of the rR spectra. Excitation with wavelengths that are in resonance with the visible electronic transitions of the azotyrosyl-248 chromophore selectively and markedly enhances the intensities of those The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Raman-active vibrational modes that are coupled to the electronic transitions. The resultant rR spectra of this enzyme reinforce conclusions based on the absorption and circular dichroic spectra obtained earlier. In addition, the behavior of vibrational bands that have now been assigned to and identified with the motions of specific atoms of the chromophoric ligand provide new information about the chemical details of the interaction of azotyrosyl-248 with the catalytic zinc atom of the active site. Work on the crystalline azoenzyme, on the interaction of the azoenzyme with inhibitors, and on model azophenols has also been completed and bands have been assigned (R. K. Scheule, H. E. Van Wart, B. 0. Zweifel, B. L. Vallee, and H. A. Scheraga, unpublished data). MATERIALS AND METHODS Carboxypeptidase A, prepared by the method of Cox et al. (6), was obtained as a crystal suspension (Sigma Chemical Co.) and purified by affinity chromatography (7). Its arsanilazo derivative was synthesized by treating a suspension of the native enzyme crystals with diazotized p-arsanilic acid as described (8). Apoarsanilazocarboxypeptidase was prepared by dialysis against 1,10-phenanthroline (9). Protein concentration was measured by the absorbance at 278 nm with E278 = 7.32 X 104 M-' cm-' for both the zinc azoenzyme and the apoazoenyme (4). Monotetraazolylazo-N-acetyltyrosine (TAT) was synthesized as described (10), and its concentration was determined with f416 = 4.39 X 103 M- cm-' (3). All rR experiments were performed under metal-free conditions. Buffers were extracted with 0. 1% Dithizon in carbon tetrachloride to remove traces of metals. Quartz sample tubes were soaked in concentrated nitric and sulfuric acids (1:1) and rinsed with metal-free water (11). The concentration of the azochromophore was about 0.06 mM in 2 mM Tris-HCI/0.5 M NaCl and 0.05 M in Na2SO4, the latter serving as an internal frequency and intensity standard. The rR spectra were obtained by using excitation from the 488.0 nm line of a Coherent Radiation argon ion laser. The power at the sample was 10-40 mW. To minimize the absorption of the incident and scattered radiations, the sample was oriented obliquely to the incident laser beam. The light scattered from the surface of the sample tube at 900 to the incident beam was focused on the entrance slit of a Spex 1401 double monochromator operated in a stepping mode. The monoAbbreviations: rR, resonance Raman; TAT, monotetraazolylazo-Nacetyltyrosine. t "Arsanilazotyrosyl-248 carboxypeptidase," "arsanilazocarboxy-
peptidase," "zinc azoenzyme," and "azoenzyme" all are terms used
interchangeably for monoarsanilazotyrosyl-248 zinc carboxypeptidase; "apoazoenzyme" is used interchangeably for apoarsanilazotyrosyl-248 carboxypeptidase. 3273
3274
Proc. Nati. Acad. Sci. USA 74 (1977)
Biochemistry: Scheule et al.
chromator and photomultiplier tube were interfaced to an Interdata 70 computer. Typically, photons were counted at each frequency for 1 min and the data were stored in digital form on magnetic tape cassettes. Spectra were plotted from these data by using programs with noise-smoothing and scale-varying capabilities. Resolution was 8 cm-1 in all cases. All band positions and molar intensities were determined relative to the 981 cm-1 symmetric stretching mode of the sulfate ion (12). Sharp bands have an accuracy of ±1 cm-1. All spectra were obtained at 200C, except that those of the apoazoenzyme were recorded at 5°C to prevent denaturation in the course of the rR experiment. Spectra obtained from consecutive scans of any given sample were reproducible, and photodecomposition was not observed. The peptidase and esterase activities of the arsanilazoenzyme were identical before and after exposure to the laser beam.
c
0 0
450
Wavelength (nm)
RESULTS AND DISCUSSION rR Characterization of Azotyrosyl-248 Species. The existence of three interconvertible species of the azotyrosyl-248 residue of the zinc azoenzyme has been demonstrated by means of absorption and circular dichroism spectroscopy (3, 5). Titration of the zinc azoenzyme from pH 6 to 11 converts the protonated azophenol first to the intramolecular azophenolate-zinc complex and, ultimately, to the free azophenolate ion (Fig. 1 upper). The vibrational bands in the 1000-1600 cm-' region of the rR spectra of the azoenzyme distinguish and characterize these three species over this same pH range (Fig. 1 lower). At intermediate pH values the rR spectra were superimpositions of these three basic spectra. Many of the rR bands of all three species are virtually the same; however, several differ characteristically and so are diagnostically valuable. These differences consist of the presence or absence of certain bands and changes in frequency or intensity, or both, of others. We shall first describe the rR characteristics of the three azotyrosyl-248 species, based on band assignments deduced from the Raman, rR, and infrared spectra of several model azophenols and their 15N and 2H derivatives. Zinc Arsanilazotyrosyl-248 Carboxypeptidase A. All three species of azotyrosyl-248 exhibit a very strong band at about 1430 cm-', due to an N=-N stretching motion. The exact frequency of this band is characteristic of the particular species present; the coexistence of more than one species of the azoprobe can be inferred from the existence of multiple bands in this region. All three species also display a band due to a C-N stretching motion at about 1389 cm-'; formation of the complex greatly enhances its intensity. In addition, all the rR spectra exhibit a doublet with bands near 1150 and 1160 cm-1 whose intensity ratio is a useful index of the species present, even though the individual frequencies remain largely independent of pH. The formation of the azotyrosyl-248-zinc complex coincides with the appearance of three sharp rR bands that are either nonexistent or very weak in the spectra of the other species. The most intense of these occurs at 1338 cm-'. It is absent at low pH and is broad and poorly defined at high pH. The weak band at 13M5 cm-' (Fig. 1 lower) is due to the small amount of the zinc complex that is present at pH 6.2.§ Another as yet unassigned band, associated only with the complex, occurs at 1212 cm-1, § The relative resonance enhancements of the three azoprobe species were found to be proportional to their molar absorptivities at 488.0 nm (see Fig. 1 upper). Hence, the small amount of the zinc azoenzyme complex present at pH 6.2 contributes measurably to the total
rR scattering observed.
pH 10.8
pH 8.5
1
3
V
386~~~~~~o I
1538
~~~~~~1216
Il LI
~~~~~~~~~~1150 1120
1486
1314
0y 7
12
pH 6.2 o456
'339~~~~~~13 ' iJ
1500
