printed in u.s.a. · 5.35 x lo4 m-’ cm-l for monkey c (20); and 5.70 x lo4 m-i cm-l for bovine b...

TIIE JOURNAL cm BIOLOGICAL CHEMISTRY Vol. 243, Ko. 17, Issue of September 10, pp. 4574-4587, 1968

Printed in U.S.A.

Carbonic Anhydrase-Azosulfonamide Complexes

SPECTRAL PROPERTIES*

JOSEPH E. COLEMAN

(Received for publication, March 26, 1968)

From The Department of Riochenaist~y, Yale Univer&, New Haven, Connecticut 06510

SUMMARY

Carbonic anhydrase combines with the optically inactive azosulfonamide (disodium Z-(4+ulfamylphenylazo)-7 - acetamido-1-hydroxynaphthalene-3,6-disulfonate) to form a highly colored enzyme-inhibitor complex. Large visible Cotton effects ([Ml = 120,000”) are induced in the multiple visible absorption bands (~600 = 25,000) of the phenylazonaphthol chromophore by the dissymmetric environment of the protein binding site. Bathochromic-hypochromic shifts of these bands accompanying binding indicate the presence of a hydrophobic binding site. Circular dichroism spectra of azosulfonamide complexes of five isozyme and species variants of carbonic anhydrase reveal that both sign and magnitude of the multiple large visible ellipticity bands ([O] = & 0.5 - 8.0 X lo4 deg cm2 per decimole, RK = f 0.4 - 19.7 X 1O-3g c.g.s.u.) are unique for each isozyme. Thus the azosulfonamides become sensitive probes for conformational features of the active center of carbonic anhydrase. Ultraviolet ellipticity bands ([e] = 1.5 x lo6 deg cm2 per decimole) induced in the phenylazonaphthol chromophore are large enough in certain isozyme complexes to completely dominate both the circular dichroism and the rotatory dispersion of the enzyme-inhibitor complexes. In accordance with the metal ion specificity observed for the induction of hydration or esterase activity in apocarbonic anhydrase, only Zn(I1) and Co(I1) at the active site induce the specific binding of the azosulfonamide which imparts optical activity to the visible absorption bands of the sulfonamide. Marked differences in the pattern of ellipticity bands induced in the sulfonamide by the &@I) and Co(I1) enzymes, however, indicate participation of the metal ion in determining the final conformation of the bound sulfonamide or the active center as well as determining the binding affinity of the enzyme for the sulfonamide inhibitors.

Sulfonamides were discovered to be potent inhibit’ors of the zinc metalloenzyme, carbonic anhydrase, during the early investi-

* This work was supported by Grant AM-09070-04 from the National Institutes of Health, United States Public Health Service.

gation of this enzyme (1, 2). The inhibition appears to be relatively specific for carbonic anhydrase, since it is the only purified enzyme reported to be inhibited by these agents (3). Studies in tivo on the inhibition of calcification by sulfonamides have implied inhibition of alkaline phosphatase (3, 4), although the crystalline zinc alkaline phosphatase from Escherichia coli does not appear to be inhibited by these agents.1 Sulfonamides have played an important role in studies of the active site of carbonic anhydrase, since sulfonamide binding has been shown to be (a) metal ion dependent (5,6), (b) accompanied by changes in energy and optical activity of the d-d transitions of the Co(I1) derivative of the enzyme (5,7), and (c) limited to the metallocarbonic anhydrases containing Co(I1) and Zn(II), the only first transition and IIR metal ions which restore significant enzymatic activity to apocarbonic a.nhydrase (6, 8).

The molecular structure of the crystalline human carbonic anhydrase C-acetoxymercurisulfrlnilamide complex at a resolution of 5.5A shows the inhibitor to be inserted into a crevice lead- ing to the zinc abom (9). The met,al atom is located near the center of the molecule. YIuch evidence now indicates that the sulfonamide group is near the metal atom and probably occupies a position within the coordination sphere (5-9). In order to be highly effective inhibitors, the sulfonamides must possess an unsubstituted sulfonamide group attached to a ring system (10). Recent data indicate, however, that certain selective modifica- tions of the sulfonamide group can be made without destroying the binding affinity completely (11). On the other hand, the ring structure can be varied widely without affecting the high affinity of the inhibitor for the enzyme (10). This latter variation allows the insertion of a great variety of chromophores into the ring port,ion of the sulfonamide inhibitors and the subsequent study of their interaction with carbonic anhydrase by the changes in absorption, optical rotatory dispersion, and circular dichroism which accompany insertion of these symmetrical chromophores into the dissymmetric environment of the active center of the enzyme. The present paper reports representative examples of these studies and demonstrates the striking changes in optical activity of the electronic transitions of a symmetric small mole-

1 Sulfonamides do not alter the visible absorption bands of Co(II) alkaline phosphatase which has a complex intense multi- banded spectrum rather similar to that of Co(I1) carbonic anhydrase (M. L. Applebury and J. E. Coleman, in preparation).

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Issue of September 10, 1968 J. E. Coleman 4575

cule that can occur on the insertion of such chromophores into a protein.

The optical activity arising from a complex chromophore such as an azosulfonamide may dominate the protein rotation in both the visible and ult,raviolet. The induced circular dichroism of the azochromophores is highly sensitive to small variations in protein structure and can easily distinguish between the several isozymes of carbonic anhydrase that have been isolated. Sul- fonamides thus become sensitive probes of the topography surrounding the act’ive site as well as providing a useful model system for the study of the optical rotatory effects of inserting a small complex chromophore into a large protein molecule. A preliminary report on some of the properties of carbonic anhydrase-azosulfonamide complexes has been given (8).

MATERIALS AXD METHODS

Reagents-All chemicals were reagent grade. Buffer solutions, HCl, NaOH, substrates, and indicators were prepared metal-free as previously described (12, 13). Spectrographically pure CoCIZ (Johnson Matthey Company, Ltd.) was used in preparing the Co(H) enzyme. Guanidine HCl was prepared from guanidine carbonate (Eastman Organic Chemicals) by the method of Ka- wahara, Kirshner, and Tanford (14).

Sulfonamides-2 - (4 -Sulfamylphenylazo) - 7 - acetamido - 1 - hy - droxynaphthalene-3,6-disulfonate was purchased from Winthrop Laboratories. 4’-Sulfamyl-2,4-diamino azobenzene was synthesized by diazotizing sulfanilamide in the usual fashion with HCl and NaN02 (15) followed by coupling with m-phenylenedia- mine under the conditions described in the literature (16). The compound showed a melting point under vacuum of 225”; literature value, 227-228” (16). 5-(p-Sulfamylphenylazo)salicylic acid was prepared as described in Pesez (17) by diazotizing sulfanilamide and coupling with salicylic acid. The compound showed a melting point under vacuum of 216”; literature value, 220” (18).

Enzymes-Human carbonic anhydrases B and C were prepared as previously reported (19). Mczcecu mulatta carbonic anhydrases B and C were isolated in crystalline form as described in Duff and Coleman (20). Bovine carbonic anhydrase B was prepared from whole bovine blood by the met’hods described for the preparation of human carbonic anhydrase by using DEAE-Sephadex chromatography (19). Cobalt(I1) carbonic anhydrase was prepared by making the apoenxymes as previously described (7) followed by dialysis against a buffered solution of Co(I1) prepared from CoCls. Protein concentrations were determined from measurements of the optical densities at 280 rnp using molar absorp- tivit,ies of 4.90 X lo4 M-l cm-’ for human B and 5.34 X lo4 M-’

cm-l for human C (21) ; 4.88 X lo4 M-! cm-’ for monkey B and 5.35 X lo4 M-’ cm-l for monkey C (20); and 5.70 X lo4 M-I cm-l for bovine B (22).

Enzymatic S&&y-Esterase activities were determined with p-nitrophenyl acetate as substrate (23) and following the absorbance change at 400 rnp or 348 rnE.1 (19,24). The reaction cuvette contained 1 X 10s3 M substrate in 0.025 M Tris, 5% acetonitrile, pH 8.0, 23”. Est’erase activities were also determined with 2- hydroxy-5-nitro-a-toluene-sulfonic acid s&one as described by Lo and Kaiser (25). The reaction mixture contained 2.5 X lo* M substrate in 0.01 M Tris-0.09 M NaCl, pH 8.0,23”. The sultone was synthesized as described by Lo and Kaiser (25), nitrated (26), and twice recrystallized from ethanol. The hydrolysis was followed by the optical density change at 410 mp with a molar ex-

tinction coefficient of 1.46 X lo4 M-’ cm-l at pH 8.0 for the phe- nolate anion of the hydroxysulfonic acid.

Optical Rotatory Dispersion-These measurements were made with a Cary model 60 recording spectropolarimeter as previously described (7). Path lengths varied from 0.01 to 1.0 cm. Slit widths were programmed to keep the dynode voltage below 0.4 kv and were 0.5 mm or less between 300 and 700 mp. In the in- tensely absorbing regions of the azosulfonamide complexes several path lengths and concent,rations were employed and the base-lines were determined with the same concentration of free azosulfonamide to avoid artifact,s from high absorpt.ions or wide slit widths. Rotation is expressed as specific rotation, [a] = a/cd, where LY = observed rotation, c = concentration in grams per ml, and d = path length in decimeters. Maximum observed rotation in the present experiments was 0.04” from 230 to 700 rnp with a maximum deviation of 10.002”. Below 230 rnp the deviation of [(Y] is &300”.

Circular Dichroism-These measurements were made with a Durrum-Jasco ORD/UV-5 spectropolarimeter equipped with CD2 attachment. Maximum deflection was =tO.O02” with a maximum deviation between runs of =tO.O0005” above 230 mp and +O.OOOl” at 215 rnp. Calibration of the instrument was performed with an aqueous solution of d-10.camphorsulfonic acid (J. T. Baker Company). eL - tn = 2.20 at 290 rnp. The slit width above 300 rnp was 0.3 mm or less. Path lengths varied from 0.01 to 1.0 cm. Base-lines were run with the azosulfonamide alone. Ellipticity is expressed as Ae = eL - tR or as molecular ellipticity, [0] = 2.303 (4500/7r) (EL - ER) with units of degree (centimeters)2 per decimole. Protein concent,rations were expressed as moles per liter and molecular ellipticity, [e], has been expressed per mole of protein rather than per mole of amino acid residue in view of the fact that the ellipticity bands of the complexes arise largely from the incorporation of 1 mole of azosulfonamide. The values can be converted to approximate mean residue ellipticities by dividing by 260 for all isozymes. Solu- tions for both ORD and CD measurements were contained in 0.025 M Tris, pH 7.5, 25”, wit’h the exception of solutions of the Co(I1) enzyme which were at pH 8.5. I’ll ORD and CD measurements made on the azosulfonamide-enzyme compleses were accompanied by blank runs over the same path lengths and on identical concentrations of the protein alone and the azosulfonamide alone in order to detect artifacts from high optical density or protein turbidity. The azosulfonamide solutions showed no optical activity and protein rotadions agreed in each case with those previously observed. With the exception of one experiment measuring the dependence of the Cott.on effects on the concentration of the azosulfonamide, the spectra of the complexes were determined on solutions containing at least 5 X 1OF M enzyme and 5 x 1OF M azosulfonamide. Roth equilibrium dialysis and kinetic measurements indicate that the dissociation con- stants for the azosulfonamide-carbonic anhydrase complexes are 1OF M or less. Thus less than 57, of the sulfonamide or enzyme is present in the uncomplexed form at the concentrations of protein and azosulfonamide employed, and these solutions have been referred to as the 1: 1 complexes.

Absorption Spectra-A Cary model 15 recording spectropho- t,ometer equipped with a slide wire for expansion to 0.1 optical density unit full scale was used to obtain the absorption spectra. All absorption spectra were recorded using the solutions employed

2 The abbreviations used are: CD, circular dichroism; ORD, optical rotatory dispersion.


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4576 Carbonic Anhydrase-Axosulfonamide Complexes Vol. 243, No. 17

-I 250

FIG. 1. CD spectra (upper curz)es) and ORD spectra (lower cuTz)es) of human carbonic anhydrase B (--) and monkey carbonic anhydrase C (- - -). Conditions: 0.025 M Tris, pH 7.5, 25”. Both samples contained 1 X lo-* M protein and path lengths were varied from 0.01 to 1 cm in order to record the complete spectra.

for the corresponding ORD or CD measurements and represent t.he optical density of these solutions over the same path length used for the latter measurements.

RESULTS

CD and ORD Spectra of Human Carbonic dnhydrase B

The CD spectrum of human carbonic anhydrase B and the corresponding ORD spectrum are shown in Fig. 1 to indicate the complex set of ultraviolet dichroic bands present in the native proteins. The data are similar to those presented previously (27) with the exception that the present CD data show considerably sharper resolution of some of the small bands. There are multiple bands, perhaps as many as eight, associated with the near ultraviolet transitions of the aromatic side chains. It is hard to be certain from inspection of the curve if all the fine structure between 270 and 300 rnp represents negative ellipticity bands, but the positions of the five minima are 293 rnp, 285 rnp, 279 rnp, 275 rnp, and 265 rnp of which the first is clearly negative. Curve fitting procedures applied to this spectrum indicate this region to consist of five negative bands for each of the isozymes as will be shown in detail for the bovine enzyme.

These are followed at lower wave length by three larger positive bands at 248 rnp, 236 rnp, and 228 mp, [0] = 1.42 X lo5 deg cm2 per decimole at 236 mp. Between 220 and 200 rnp there is a large asymmetric negative band, [0] = -8.2 x lo5 deg cm2 per decimole at 211 mp. The most negative region appears centered in this preparation of the enzyme near 211 rnp with a shoulder in the 215 rnp region. The latter t,wo bands appear to be largely responsible for the trough at 222 rnp in the ORD spectrum. High absorbance of the protein prevents CD readings further in the ultraviolet, but there must be at least one addi-

tional large negative band to account for the large trough in the ORD at 196 rnp. The multiplicity and signs of the various bands from 228 to 330 rnp are not readily apparent from the ORD spectrum. The same complement of CD bands appears to be present in the other isozyme and species variants of carbonic anhydrase (see below), but their rotatory strength and precise position show considerable variation as shown by the CD and ORD of the monkey C isozyme in Fig. 1.

ORD Spectra of Carbonic Anhydrase-Azosulfonamide Complexes

Variation with Zsozyme-Of the numerous sulfonamides inves- tigated, the one showing the most striking changes in the absorption and optical rotatory properties of its absorption bands on forming carbonic anhydrase complexes is a phenylazonuphthol derivative of sulfanilamide, 2-(4.sulfamylphenylazo)-‘i-acetamido-l-hydroxynaphthalene-3,6-disulfonate (Compound I, Ta- ble I). On the binding of one molecule of the optically inactive Compound I to any of the five species or isozyme variants of native Zn(I1) carbonic anhydrase, a series of Cotton effects is induced in the visible wave length region corresponding to the absorpt,ion bands of the sulfonamide (Fig. 2). The largest, centered between 490 and 525 rnp, depending on the isozyme, has an amplitude of approximately 120,000”, expressed as molecular rotation. This induction of optical activity is accompanied by changes in the extinction coefficient and wave length of the sulfonamide absorption bands; changes which are again variable with the isozyme. The most marked change is the hypochro- micity and bathochromic shift of the principal absorption band induced by human carbonic anhydrase 13. While each isozyme has a distinctive spectral and ORD profile for it,s complex, the two C isozyme complexes are the most similar in terms of their spectral and ORD profiles (Fig. 2).


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TABLE I

4577

Sulfonamides used for spectral studies of carbonic anhydrase

structure Name

4’-Sulfamylphenyl-2-axo-7-acetamido-l-hydroxynaphthalene-3,6-disulfonate (Neo- prontosil)

4’-Sulfamyl-2,4-diaminoazobenzene (Prontosil)

5(p-Sulfamylphenylazo)salicylic acid (Lutazol)

hlcentra- tion

inhibiting esterase

ctivity .5OY,

x 106 M

1

0.3

0.1

In the ultraviolet region the differences between the conformation of the azosulfonamide bound to the various isozymes are even more marked (Fig. 3). In this regard, the monkey isozyme B complex stands out in that there appears to be a large negative Cotton effect associated with one of the near ultraviolet azosulfonamide bands. This Cotton effect is not induced by t’he human isozyme 13. Detail of the ORD in the aromatic region of the complexes is imprecise because of the high optical density. This detail is clearer in the CD spectra to be shown below.

Visible Absorption Spectrum of A zosulfonamide as Function of Solvent and pH

The absorption bands of aromatic azocompounds are known to be sensitive to the polarity of the solvent and to proton equi- libria involving the side chains as well as the diazo linkage. Spectra of Compound I in solvents of varying polarity and in aqueous solution at several hydrogen ion concentrations are shown in Fig. 4. In non-polar solvents, the main absorption band undergoes a hypochromic-bat.hochromic shift similar to that induced by carbonic anhydrase. This shift is most marked in the most non-polar solvent, dioxane; and is very sensitive to the amount of water in the dioxane. The compound is practi- cally insoluble in 1007, dioxane, but becomes very soluble upon the addition of 1 to 2% water. The relatively more polar solvent,s methanol, ethanol, and acetonitrile produce similar but less marked shifts. The absorption envelope of the compound is unchanged between pH 2 and 9. Above pH 10 it undergoes a broadening and a hypochromic shift accompanying the loss of a proton from the sulfonamide group. Titration data show the proton to titrate above pH 9.0. In 50% aqueous sulfuric acid the conjugate acids of the phenylazonaphthols are formed (28). Under these conditions the spectrum of Compound I shows a pronounced hyperchromic shift and a clear split of the main peak into two bands.

CD Spectra 0s Carbonic Anhydrase-Azosulfonamide Complexes

Variation with Isozyme-The overlapping contributions to the ORD from all absorption bands makes it difficult to sift out the

I I I (+I I AHB

A. InkI

FIG. 2. Cotton effects and spectral shifts induced in the visible absorption bands of Compound I accompanying binding to carbonic anhydrase isozymes. Upper spectra, ORD spectra of the 1:l complexes of Compound I with human isozyme B (HB); monkey isozyme B (MB); human isozyme C (Hc); monkey isozyme C (MC) ; and bovine isozyme B (B) The plain dispersion curves of all of the isozymes alone fall within the deviations indicated on the plain curve shown in the figure. Lower spectra, visible absorption spectra of the 1:l complexes of Compound I with isozymes of carbonic anhydrase. -, free Compound I; human isozyme B, __ -; monkey isozyme B, .... ; human isozyme C, -.“-; monkey isozyme C, -‘.-; bovine isozyme B, -.-. All enzyme solutions contained 5 X 1OV M enzyme, 5 X 10e5 M azosulfonamide, and 0.025 M Tris, pH 7.5, 25”.


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6-

9-

12- I I

220 240 260 260 300 320

FIG. 3. Ultraviolet ORD spectra of carbonic anhydrase isozymes and their 1:l complexes with Compound I. Spectra of the isozymes alone are indicated by Hg, human isozyme B; MB, monkey isozyme B; H C, human isozyme C; MC, monkey isozyme C; B, bovine isozyme B; C’s, 1:l complexes. Spectra of the complexes are indicated by the same designations + I; e.g., HB + I, the complex of human isozyme B. All solutions contained 5 X lop5 M enzyme (+5 X 10m5 M azosulfonamide in the case of the complexes), 0.025 M Tris, pH 7.5, 25”. All were recorded over a 0.2-cm path length.

effect in the ORD of the monkey R isozyme (Fig. 3) is shown to be made up of a negative band at 254 rnp, [0] = -1.5 X lo5 deg cm2 per decimole and a positive one near 228 rnp, [0] = + 1.5 x lo5 deg cm2 per decimole. Both are of sufficient magnitude to completely change the character of the ultraviolet ellipticity bands of the complex as compared to those of the protein alone.

These structural differences indicated to be present in the active centers of the various isozymes by the CD spectra of the azosulfonamide complexes are also reflected in function as shown by the esterase activities of these species and isozyme variants (Table II). Two esters representing extremes of structural variation were chosen, p-nitrophenylacetate and 2-hydroxyd- nitro-oc-toluenesulfonic acid s&one. The latter can be regarded as a substrate analogue of the sulfonamides (25).

ORD and CD Spectra of Human Carbonic Anhydrase B-Azosul- fonamide Complexes as Function of Enzyme

Preparation and Metal Ion

The ORD spectrum of a fresh solution of lyophilized human sozyme B plus equimolar azosulfonamide is shown by Curve 1

LI I I I I I --I

number of Cotton effects or optically active absorption bands present in a spectrum as complex as that of an azosulfonamide- protein complex. The CD spectra of two representative isoayme complexes, monkey B and human C, are shown in Fig. 5. The CD of the monkey C isozyme complex is almost identical with that for the human C isozyme. The positions of prominent bands are marked on the CD spectra indicating the rather considerable variation between isozyme complexes in the position of these main bands, as much as 14 rnp for the largest visible band. The ultraviolet CD of the native monkey isozyme B and human isoayme C indicate t’he same set of optically active transitions to be present in both enzymes. Position and rotatory strength, however, show considerable variation which must reflect structural differences; e.g. the shift of the first large positive band from 236 rnp in the B isozyme to 245 rnp in the C isozyme and ellipticities of $1.1 x lo5 and +4.5 X lo4 deg cm2 per decimole, respectively.

Marked differences in the CD spectra of their respective azosulfonamide complexes indicate considerable differences in structure around the active sites of these two isozymes. Particularly striking features are the large ultraviolet dichroic bands induced in the sulfonamide chromophore by monkey enzyme B and missing in the C isozyme. What appears as a large negative Cotton

t A

i :

‘~50% Aqueous H,SO, - L i

0 I I I I 350 400 450 500 550 600

X (m,ul)

FIG. 4. Absorption spectrum of azosulfonamide I as a function of solvent and pH. A, Compound I, 5 X 10e5 M, was dissolved in Hz0 (0.025 M Tris, pH 7.5, 25’) (-); methanol, 99% (-..-); ethanol, 95% (- - -); acetonitrile, 98% (-.-); and dioxane, 96% (----). B, Compound I, 5 X lo-+ M, was dissolved in 0.025 M Tris, 25’; pH 2 to 9 (---); pH 10.2 (-- ---) ; pH 11.3 (-.-); and in 1: 1 H,SOd-HZ0 (- - -). For the high pH values, the presence of Tris was sufficient to prevent any rapid changes in pH due to CO2 hydration. In the regions outside the major buffering region of Tris, the pH of the sample was checked before and after the recording of the spectrum.


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in Fig. 6. I f the azosulfonamide is added to the solution of the enzyme after the enzyme has stood in solution at 4” for 7 days, Curve 2 is obtained. Changes continue in the ORD spectrum of the complex for 1 to 2 weeks depending on the preparation. A stable profile is finally achieved as shown by Curve 5. This change is not reflected in detectable alterations in either the esterase or COZ hydration activities. All of the other four isozyme and species variants of carbonic anhydrase examined demonstrate completely stable azosulfonamide complexes as judged by following the time course of the ORD profiles. The change does not represent a falling dissociation constant for the complex, since excess sulfonamide does not restore the original magnitude of the visible Cotton effects. Neither does it represent changes in the azosulfonamide molecule, since successive complexes were formed with fresh azosulfonamide solutions.

The visible Cotton effects of the azosulfonamide complex can be used to determine the dissociation constant for the complex. The procedure is illustrated in Fig. 7 for the monkey isozyme B showing a plot of the visible ORD spectrum of the complex as a function of the concentration of free azosulfonamide present in the solution. Samples were prepared by equilibrium dialysis of 5 X 10-S M enzyme in closed 2-ml dialysis bags against 100~ml

t-1 I$ 15 , I I 1’ I I I I 1

200 300 300 400 500 (-)

h,mp

c-11 , , 1, , , , , , / 415

200 250 300 400 500 600

FIG. 5. CD spectra of Macaca mu2atta carbonic anhydrase B (MB) and human carbonic anhydrase C (Hc) and their 1:l complexes with Compound I. -, monkey isozyme B; - - -, monkey isozyme B plus equimolar Compound I; --, human isozyme C; - - -> monkey isozyme C plus equimolar Compound I. All solutions contained 5 X low5 M enzyme (+5 X 10m5 M azosulfonamide in the case of the complexes), 0.025 M Tris, pH 7.5, 25”.

TABLE II Esterase activities of species and isozyme variants

of carbonic anhydrase

I Esterase activity

ISOZylW3 +Nitrophenyl

acetatea Z-Hydroxy-j-nitro- a-toluenesulfonic

acid sultan@

Bovine B .............. Human B .............. Monkey B ............. Human C .............. Monkey C .............

. .

.

nzoles substrate hydrolyzed/min/mole ‘3fZ~plZ~

27.5 xk 4.7 1253 f 104 11.5 i 0.5 658 f 14 70.1 f 7.9 530 f 10 50.8 f 3.2 1150 + 50 46.S f 0.8 750 f 15

5 Conditions: 0.025 PA Trist pH 8.0, 23”. b Conditions: 0.01 M Tris-0.09 M NaCl, pH 8.0, 23”.

(3

(+I

12- n -

X.mp

FIG. 6. Change in the ORD spectrum of the I:1 complex of human carbonic anhydrase B with Compound I as a function of time. A solution of 5 X 1OV M lyophilized human carbonic anhydrase was made up at zero time. A fresh solution of 5 X IO+ M Compound I WBS then added at various time intervals and the ORD spectrum recorded; Curve 1, zero time; Curve 2, 1 week; Curve 3,3 weeks. The plain dispersion curve is that for the human isozyme B at zero time or 3 weeks. - - -, ORD spectrum of the 1:l complex of Co(I1) human carbonic anhydrase B with Com- pound I. The solution contained 5 X 1O-5 M Co(I1) enzyme and 0.025 M Tris, pH 8.5, 25”.

volumes of the azosulfonamide solutions. The method is similar to that described in Reference 29. Final concentrations of free azosulfonamide were calculated by correcting for the protein- bound dye extracted from the medium or from the absorbance of the dialysate at 500 rnp (6 = 25,000). The Cotton effects are half-developed at -1.5 x low6 M free sulfonamide. This value agrees well with the order of magnitude indicated for Ki by preliminary data with the azosulfonamide to inhibit the esterase activity of the monkey isozyme B (Table I). Addition of the azosulfonamide to an enzyme treated with 2 M guanidine HCI either before or after the addition of the sulfonamide does not generate the Cotton effects seen with the native enzyme (Fig. 7).

While the ORD curves in Fig. 6 indicate a considerable difference in the binding characteristics of the azosulfonamide to the human isozyme B, the ellipticity bands shown in Fig. 8 indi-


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4580 Vol. 243, No. 17 Carbonic Anhydrase-Axosulfonamide Complexes

-

FIG. 7. Visible Cotton effects induced in azosulfonamide I by monkey carbonic anhydrase B as a function of the concentration of free azosulfonamide and in the presence of 2 M guanidine HCl. - -, ORD of 5 X 10m5 M enzyme; Curve 1, plus 6.7 X 10-s M Compound I; Curve 9, plus 5 X 10e7 M Compound I; Curve d, plus 7.7 X lo-’ M Compound I; Curve .J, plus 8 X 10e6 M Com- pound I; Curve 5, plus 1 X lo+ M Compound I; Curve 6, plus 2 X 10e6 M Compound I. Concentrations of Compound I for Curves 1 to 6 refer to the concentration of unbound Compound I. -.-, ORD of 5 X low5 M enzyme, plus 5 X low5 M Compound I plus

cate that, the final conformation of this complex deviates in a qualitative fashion from the other isozyme complexes. The ellipticity bands associated with the azosulfonamide transitions in the human enzyme B complex are both positive and negative and do not correspond in any simple way to the ellipticity bands present in t.he monkey isozyme B or human isozyme C as judged by simple inspection of the CD envelopes (Fig. 5).

The ORD of the Co(I1) human isozyme I%azosulfonamide complex was included in Fig. 6. This should be compared to Curve S for the Zn(I1) enzyme complex, since it takes at least 15 days of dialysis to prepare the Co(I1) enzyme. The ellipticity bands of the two complexes are compared in Fig. 8. The main positive ellipticity band above 500 rnp moves from 548 rnp in the Zn(I1) human enzyme B complex to 506 rnp in the corresponding Co(I1) complex accompanied by the disappearance of the negative ellipticity band at 476 ml* present in the Zn(I1) enzyme complex. While some of this difference may be related to the super- imposition of ellipticity bands arising from the d-d transitions of the Co(I1) ion, the magnitudes of these bands as observed in the complexes of the cobalt enzyme with colorless sulfonamides (30) have not been large enough or of the right sign to produce such marked changes in the ellipticity band pattern. The azosulfonamide does induce a shift in the position of the d-d transi-

2 M guanidine-HCl and 0.025 M Tris, pH 7.5. Inset, percentage maximum amplitude of the peak at 540 rnp (0) or the trough at 440 rnp (B) plotted as a function of free azosulfonamide concentration. % MAX. AMPLITUDE f re ers to the difference in degrees at the given wave length between the plain CUTY~ and the numbered curve divided by the difference between the plain curve and Curve 6 expressed as a percentage. The midpoint of this function occurs at 1.5 X low6 M free azosulfonamide and can be interpreted as the dissociation constant for the enzyme-inhibitor complex. The right-hand ordinate should read observed rotation (deg X 10%).

tions of the enzyme-bound Co(I1) ion as shown by the difference spectrum in the lower part of Fig. 8. This difference spectrum was obtained by recording the spectrum of a 2.16 X 1O-4 M solution of the Co(I1) enzyme-azosulfonamide complex versus an equimolar concentration of the Zn(I1) enzyme azosulfonamide complex. The intense absorption of the azo dye is thus removed leaving the d-d transitions of the Co(I1) ion. This difference spectrum is compared to the d-d absorption bands of the Co(I1) human isozyme B alone and demonstrates the spectral shift characteristic of the combination of the Co(I1) enzyme with colorless sulfonamides (5, 7, 30).

Gaussian Resolution of Visible CD Spectra of Human B and Bovine B-Azosulfonamide Complexes

The CD spectra of the human B and bovine B isozyme-azosulfonamide complexes show the greatest variation in the general contours of their visible ellipticity bands (Figs. 8 and 9). In order to demonstrate more specifically what this variation means in terms of band structure, both CD spectra have been resolved into their component gaussian bands by means of the DuPont 310 curve analyzer (31, 32) (Fig. 9). Since the gaussian nature of absorption bands relates to a gaussian distribution of energies, the spectra have been plotted as a function of wave number for


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purposes of curve fitting. The deviation of a fitting based on wave length from one based on frequency is considerable in the case of these broad bands. Resolution of the CD spectrum of the bovine complex (Fig. 9A) into a set of gaussian ellipticity bands is detailed in Fig. 9B, while the resolution procedure applied to the CD spectrum of the native enzyme is shown in Fig. 9C. Two alternative sets of gaussian bands for the low energy end of the CD spectrum of the complex are presented in Fig. 9B.

These will be discussed in connection with the computer calculation of the corresponding ORD spectra. The positions, molecular ellipticities, half-band widths, and calculated rotatory strengths for each of these bands are given in Table III.

Of the ten gaussian bands required to fit the CD of the bovine complex, four are small negative bands (6, 7,8, and 9 in Fig. 9B)

which fall in the region of the small aromatic bands of the native protein. This set of bands appear to represent the superposition of the aromatic bands of the protein on those of the azosulfonamide chromophore. This conclusion is supported by the appear- ance of a set of four negative bands in almost identical positions in the resolution of the CD spectrum of the native protein (Bands

6’, 7’, 8’, and 9’ in Fig. 9C). In addition, the native protein shows a fifth small negative aromatic ellipticity band (Band 11

in Fig. 9C). This complement of five negative aromatic bands appears to be a constant feature of all isozymes examined thus far (see Fig. I), although they vary considerably in relative magnitude between isozyme or species variants of carbonic anhydrase.

6 - \ 0 I ‘\.I’ \ -

300 400 500 600 700

A,mp FIG. 8. Visible CD and absorption spectra of the 1:l complex

of Co(I1) human carbonic anhydrase B with Compound I. Com- parison to the CD of the 1:l complex of Zn(I1) human carbonic anhydrase B with Compound I. Upper spectra: - - -, CD of the Co(I1) enzyme complex; --, CD of the Zn(I1) enzyme complex. All solutions contained 5 X 1OP M enzyme, 5 X 10W6 M Compound I, 0.025 M Tris, pH 7.5 (8.5 for the Co(I1) enzyme). 25”. Lower spectra: -.-, absorption spectrum of 2.16 X low4 M Co(I1) enzyme; - - -2 absorption spectrum of 2.16 X 10m4 M Co(I1) enzyme complex minus 2.16 X 10m4 M Zn(II) enzyme complex.

U x 10v4 (cm-‘) 4.6 3.6 2.6 1.6

IO -

(+I A* n

k:j: \!J iI, 1 17 4.6 2.6 1.6

V x 10e4 (cm-‘)

FIG. 9. CD spectra of bovine carbonic anhydrase B and its 1:l complex with Compound I. Resolution into a set of overlapping gaussian ellipticity bands. A: --, observed CD spectrum of the complex; - - -, observed CD spectrum of the native enzyme. B (complex) : -, resultant envelope for the ten gaussian bands labeled from 1 to 10; - - -, bands attributable to the sulfonamide; -, bands attributable to the protein. The subset curve, Bands 1’ and .9’, show an alternate fitting of the lowest energy region. C (native enzyme): -, resultant envelope for the seven gaussian bands labeled on the figure. In B and C, the envelopes correspond within the limit of error to the experimental CD curves.

In addition to the absence of Band 11 in the CD of the sulfonamide complex, there are some small changes in magnitude of Bands 6, 7, 8, and 9 compared to the corresponding bands in the native protein (Table III). This may reflect inaccuracies of the data in this region of high absorbance, particularly in the case of the complex, but slight changes in the conformation of the aromatic residues of the protein induced by the inhibitor cannot be ruled out. There are some indications from data derived from studying the binding of a fluorescent sulfonamide that interaction between the sulfonamides and the aromatic residues does occur (33).

In order to fit the CD spectrum of the native protein, the large additional ultraviolet bands at 234 rnp (Band 12) and 218 rnp (Band 1s) have to be added (Fig. 9C). No precise fitting has been attempted for the latter two bands. Like the human and monkey isozymes shown in Fig. 1, close analysis of these bands shows three components for the first and two for the second.


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TAELE III Computer Calculation of ORD for Human B and Bovine B Carbon& Band position, molecular ellipticity, and rotatory strengths for Anhydrase-Azosuljonamide Complexes

resolved gaussian. components of CD spectra of bovine and human carbonic anhydrase B-azosulfonamide complexes

The argument can be advanced that a curve fitting procedure of the type presented above does not produce a unique solution

Band position Molecular Half- band

of overlapping gaussian functions, a problem which has been Band

llipticity: width=

Rotatory

OX &I x A; X itrengw

0 XK 10-4

i discussed previously (32). The validity of t,he curve fitting

9 !K x loas lo-” 10-a procedure can be checked to a certain degree by using the param-

eters derived from the gaussian ellipticity bands to calculate the +w cm-’ deg cm=/

d&mole cm-’ cgs u resultant ORD spectrum. A set of gaussian ellipticity bands

Bovine carbonic anhy- which satisfies the CD data and generates the experimentally ob-

drase B-azosulfon- served ORD spectrum can be regarded with more confidence than amide complex a fitting of the CD spectrum alone. Fitting of a visible ORD

1’ 505 1.980 6.68 0.180 7.49 spectrum, however, may present some additional problems be- 2' 453 2.210 2.25 0.157 1.97 cause of large contributions from ellipticity bands at lower wave 3 386 2.590 2.29 0.270 2.94 lengths. 4

The problems are well illustrat.ed by the two examples 338 2.960 2.11 0.390 3.43 shown here.

5 290 3.460 8.78 0.630 19.72 The procedure consisted of selecting those gaussian

6 296 3.380 -2.11 0.190 -1.46 ellipticity bands from Figs. 9 and 10 which can be clearly assigned

7 283 3.540 -2.54 0.070 -0.62 to the sulfonamide (Bands 1, 2, 5, 4, 5, and 10 in the bovine en-

8 275 3.635 -1.89 0.080 -0.51 zyme complex and I,!?, S, 4, and 5in the human enzyme complex).

9 267 3.750 -2.64 0.140 -1.22 The partial rotation arising from each of these bands was then

10 267 3.750 3.21 0.240 2.53 calculated by evaluating the Kronig-Kramers transform relating

Native bovine carbonic absorption and dispersion phenomena (34). The expression anhydrase B relating rotatory strength a?d the parameters describing the

6' 296 3.380 -1.43 0.190 -0.99 ellipticity bands (Table III) to the molecular rotation is given by 7’ 284 3.524 -1.78 0.070 -0.44 8’

Equation 1 (34). The integral for each band as well as the sum 276 3.620 -1.96 0.080 -0.53

9’ 267 3.740 -2.86 0.140 -1.32 11 260 3.840 -1.64 0.115 -0.61

e-(v-v;)2/( A;;)”

12 234 4.240 11.07 0.400 12.88 (1)

Human carbonic anhy- s

(v-n;,/A> A:

drase B-azosulfon- ez2 dx - 2(Y + &)

amide complex II 1

1 544 1.825 1.74 0.071 0.84 for all bands was evaluated by means of a computer program 2 508 1.938 0.75 0.053 0.25 written for the General Electric 235 computer, substituting 3 457 2.090 -1.20 0.164 -1.16 4 X60 2.763 3.34 0.308 4.60 5 292 3.434 6.27 0.240 5.41 (+) 1 6 296 3.389 -2.06 0.056 -0.75 - 7 287 3.483 -2.53 0.057 -0.93

4- 8 278 3.488 -3.74 0.113 -2.65 9 259 3.850 4.84 0.150 4.10

L


a RK = Im [ &rni cos e)], loci = electric dipole moment of tbe ith 3- transition, rni = magnetic dipole moment of the ith transition, t

0 = angle between the vectors ra and me; RK was calculated with h the expression, Rg = 0.696 X 10P2 & [&] A;/& A; = x half-band widt,h = interval between [0:] and (l/e) [s”,] (see Moscowitz (34) for derivation and definition of terms). s 0

A similar set of major azosulfonamide ellipticity bands is required to fit the CD spectrum of the human isozyme B complex (Bands 1 to 5, Fig. lo), but magnitude and sign as well as posi- 3-

tion of the bands are changed (Table III). Of all the bands, only Band 5 seems to correspond closely to its counterpart in the (-) bovine enzyme complex. Bands 6, 7, and 8 (Fig. 10) are prob-

, v

ably enzyme bands arising from the aromatic chromophores (see 4.6 3.1 1.6

Fig. 1). The full complement of negative aromatic ellipticity Y x 10v4 (cm-‘)

bands, however, does not appear in the complex. Again this may be poor resolution in this area of the CD spectrum or an altera-

FIG. 10. Resolution of the CD spectrum of the 1:l complex of Zn(I1) human carbonic anhydrase B with Compound I into a

tion in these bands induced by the inhibitor. Band 9 cannot be set of overlapping gaussian ellipticity bands. -, resultant

interpreted since both protein and azosulfonamide chromophores envelope for the nine gaussian bands indicated by the dashed curves

are overlapping here and the gaussian analysis of the native pro- and labeled 1 to 9. The envelope corresponds within the limit

tein has not been carried this far into the ultraviolet. of error of the measurement to the experimental CD spectrum of the complex (see Fig. 8).


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values of v at intervals of 500 cm-l from 1.0 to 4.0 X lo4 cn-I. As in the analysis of the CD spectrum, the integral in Equat,ion 1 has been expressed and plotted in terms of frequency rather than wave length. The computed rotatory dispersion was then added to the plain rotatory dispersion of the protein alone and compared with experiment.

The ORD of the human isozyme B-azosulfonamide complex proved to be the most readily fitted by this procedure using the set of ellipticity bands shown in Fig. 10. The individual computer-generated Cotton effects resulting from Bands 1 to 5 are shown in Fig. llA, while the sum of these Cotton effects is

plotted in Fig. 1lB as Curves 1 to 5. This curve when added to the plain rotation of the protein generates a curve of similar shape to the one observed for the azosulfonamide complex. Rotation throughout the visible region, however, is more positive than actually observed. The most likely explanation would seem to be that there are significant negative contributions from

+3

+2

+1 *

b -0 x

% -1

-2

-3

t4

t2

d I QA

x

FJ

-2

-4

-6

-8

r

i

f c

I I I I I I I I I I I I I I I I I I I

lll,lllllllllllllll1.J I/

3.4 2.4 1.4

r) x 10e4 (cm-‘) FIG. 11. Computer calculated ORD of the human carbonic

anhydrase B complex with Compound I. A, Curves 1 to 5 are the individual Cotton effects generated by ellipticity Bands 1 to 5 in Fig. 10. Curve 6 is an arbitrary negative Cotton effect fed into the computer to adjust for ultraviolet contributions to the dispersion (see text). B: - - -, plain visible dispersion of human isozyme B; p, experimental ORD of the complex (3 weeks in solution). Curves 1 to 5 and 1 to 5 + 6 are the sums of the corresponding individual Cotton effects in A. l , Curve 1 to 5 + 6 added to the plain rotation of the protein.

$8 I-

0

x

G

t6-

-6- /

/ I I I I I I I

3.4 2.4 1.4

2, x 10e4 (cm-‘)

FIG. 12. Computer calculated ORD for the bovine carbonic anhydrase B complex with Compound I. - - -, plain visible dispersion of bovine isozyme B; -, experimental ORD of the complex. Curves 1, 2, 3, 4, l’, Z’, 3, 4, and l’, Z’, 3, 4, 5, 10 are the sums of the Cotton effects generated by the corresponding ellipticity bands in Fig. 9. l , Curve l’, Z’, 3,4 added to the plain rotation of the protein.

ultraviolet ellipticity bands induced in the azosulfonamide by the

protein. At least two such bands appear in the monkey isozyme B complex (Fig. 5). In general the other complexes have smaller near ultraviolet bands or much larger deep ultraviolet bands which are not accessible to measurement because of the high absorption of these complexes. A reasonably good fit of the computer-generated ORD to the experimentally observed curve can be achieved by arbitrarily adding a single negative dispersion curve to compensate for t’he ultraviolet contributions (Curve 6 in Fig. 1lA). By altering the position and magnitude of t’his band, the fitting shown by the points in Fig. 1lB was obtained. There is slight overcompensation in the region of 3 X lo4 cm-’ and a deviation around 2.1 x lo4 cm-l. The latter may result from changes in the protein sample between the ORD and CD measurements, since this complex undergoes alterations in the visible ellipticity with time (Fig. 6).

Similar calculations applied to the bovine isoayme B-azosulfonamide complex reveal some additional features of t,his type of analysis (Fig. 12). The observed ORD spectrum for this complex can be closely fitted in the visible region by the procedure

described above using only the four lowest energy bands of the CD spectrum (Curve 1’, .Z’, S, 4 in Fig. 12). In addition, only one of the two alternate fittings of t,he CD spectrum in Fig. 9B generates the ORD curve. These two alternate fittings, however, both fit the CD spectrum wibhin the experimental error of the measurement. Because of the narrowness of the large visi-

ble ORD peak, the low energy band of the CD spectrum must be accounted for by one large band (Band 1’, Fig. 9B). The alternate fitting of two equal sized bands generates a broad peak with


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Carbonic Anhydrase-Axosulfonamide Complexes Vol. 243, No. 17

much larger positive values of rotation than observed experimentally (Curve 1, 2, S, 4 in Fig. 12). The successful four-band fitting added to the plain rotation of the protein is shown by the points in Fig. 12. If the positive CD bands (5 and 10) are added to the analysis, the proper shape of the ORD is maintained, but like the initial fitting of the human enzyme B complex, there is a marked positive deviation throughout the visible. In view of the fact that the four-band fitting is very close to the experimental ORD in the visible region, the effect of the large positive ellipticity Band 5 on the visible ORD must be effectively can- celed by negative bands further in the ultraviolet. This can- cellation is also implied by the observation that the experimental ORD for the bovine complex as shown in Fig. 3 deviates only moderately from that for the native protein in the region of Band 5, 270 to 350 mp. Addition of a single negative band to compensate for such contributions is not as successful, however, as with the smaller correction needed for the human enzyme complex. The true contribution must reflect the composite dispersion curve from a number of overlapping ultraviolet ellipticity bands. It can be seen from the data in Fig. 12 (Curve 1’, g’, S, 4, 5, 10) that these ultraviolet contributions to the dispersion in the visible must be equal to or greater than those of the intrinsic chromophores of the protein.

Optical Activity of Carbonic Anhydrase-Azosulfonamide Complexes as Function of Azosulfonamide Xtructure

Since the absorption spectra of the azobenzenes and azonaphthols are known to be extremely sensitive to the nature of the ring structure and the side chains, the effects of these variables on the optical activity induced in the azosulfonamide absorption

lo- 1 11 I_ (+I i!

P 0 -

x

E

c-1 0 x

0 CL

h (mpu) FIG. 13. CD and absorption spectra of 1:l monkey carbonic

anhydrase B-azosulfonamide complexes as a function of azosulfonamide structure. CD (upper curves): -, enzyme plus Compound I; - - -, enzyme plus Compound II; -.-, enzyme plus Compound III. Absorption spectra (lower curves): the spectra of the bound sulfonamides are given by the symbols corresponding to the CD spectra above; -, Compound I; - - -, Compound II; -.-, Compound III. The spectra of the free compounds are given for comparison; . . . ., Compound I; -, Compounds II and III.

bands by carbonic anhydrase were examined. Two phenyl- azosulfonamides (Compounds II and III in Table I) were synthesized. Both inhibit carbonic anhydrase as effectively as Compound I (Table I). The visible absorption spectra of the free compounds, the compounds bound to monkey carbonic anhydrase B in a 1:l molar ratio, and the induced ellipticity bands of the bound compounds are shown in Fig. 13 and compared to the analogous spectra for Compound I. The most striking features are the radical differences in the absorption spectra of these three azosulfonamides induced by changing the side chains or the ring structure. Compound III has a band at, -434 rnF which shows the greatest induced ellipticity, even though it is a weak absorption band on the edge of the intense band at 355 rnp. The latter intense band, however, does not have much induced rotatory power. If the carboxyl and hy- droxyl groups in the meta and para positions of Compound III are replaced by amino groups in the ortho and para positions (Compound II), the long wave length bands which still show the major induced rotatory power, gain some intensity, while the very intense bands of the spectrum move to much lower wave length without affecting the general pattern of the induced ellipticity bands.

DISCUSSION

Primate erythrocyte carbonic anhydrases from species examined thus far have been shown to be present as at least two isozymes (20, 35-37). In those instances where the isozymes from a single species have been separated and isolated as homo- geneous preparations, the amino acid compositions show considerable variation between isozymes (19, 20, 22). Bovine and canine erythrocyte carbonic anhydrases on the other hand do not show such clear evidence for isozymes differing in amino acid composition. The latter do differ considerably in amino acid composition from the primate isozymes (22, 38). In spite of significant differences in amino acid composition, there are a number of general physicochemical parameters such as molecular weight, zinc content, ORD, and CD spectra (Fig. 1 and 2) (7, 20, 27, 39), visible absorption spectra of the Co(I1) deriva- tives (5, 20, 30), and high affinity for sulfonamides which indicate that certain gross structural features of the molecule must be common to the whole series of isozyme and species variants. There are a number of more subtle indications, however, that there are some specific differences between these various carbonic anhydrases, presumably reflect’ing substitution of certain amino acid residues. The esterase activities of these isozymes vary markedly, both in magnitude (Table II) and in the position of the midpoint of the pH rate profiles (24). The details of the aromatic Cotton effects, characteristic of these molecules, also vary between isozymes (Fig. l), implying different environments for the aromatic residues. Binding affinities for the sulfonamides, while relatively high for all mammalian carbonic anhydrases, change as a function of isozyme implying some structural differences between the binding sites

(10). The observation that multiple chromophores of the enzyme-

bound hydroxynaphthalene derivative of an azosulfonamide show significant optical activity, clearly induced by the surrounding dissymmetric protein structure, suggested that this induced optical activity might be extremely sensitive to variations in the protein structure at or near the binding locus. This proved to be the case, and since the evidence is now overwhelming that


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the sulfonamides do bind very near the metal atom at the active center of carbonic anhydrase (5, 6, 9), this dye becomes a sensitive probe for variation in structure at the active centers of the several isozyme and species variants.

The pairs of B and C isozymes from human and monkey erythrocytes have appeared to be closely related. Amino acid compositions are nearly the same for the respective isozymes (20) as are the ORD patterns (7, 20, 27, 40). Probing the structure of the active center with the azosulfonamide does not change these conclusions in the case of the C isozymes. The spectral shifts induced in the azochromophore and the contours of the visible ORD and CD of the complexes are very similar (Figs. 1,2, 3, and 5). In contrast the azosulfonamide detects a marked difference between the sulfonamide binding sites of the human isozyme B and the monkey isozyme B. The CD spectra (Figs. 5 and 8) indicate not only large differences in induced rotatory strength, but that the conformations of the bound azosulfonamide are sufficiently different so that certain of the helical paths of the electrons involved in the visible transitions have reversed their helical sense from right to left. In the ultraviolet, the very large ellipticity bands induced in the sulfonamide chromophore by the monkey isozyme B are missing in the human isozyme B complex. It can of course be argued that a single properly placed amino acid substitution could force this difference in the conformation of the bound sulfonamide. Esterase activities also indicate functional differences between the active sites of the various isozymes; the most marked difference occurs in the rate of hydrolysis of p-nitrophenylacetate by the two B isozymes (Table II).

An additional difference between the B isozymes is brought out by the change with time in the ORD of the human enzyme B complex as it stands in solution (Fig. 6). This is a function of the protein structure alone (see “Results”) and must reflect a subtle change in the surface structure of the molecule. It is of interest that of all the isozymes only the human enzyme B will not crystallize readily from solution. This could be related to a small conformational change in the human isozyme B that con- tinues in solution and is detected by the change in the azosulfonamide Cotton effects. I f crystallization of this isozyme is carried out in the presence of a sulfonamide, crystals are readily obtained.3

The great sensitivity of the intensities and induced ellipticity of the azosulfonamide absorption bands to small variations in the structure of the inducing protein provide some significant information on the mechanisms involved in binding. The first striking observation is that the rotatory power, RK = Im (pimi cos 0) (34), of these induced dichroic bands approaches 1O-38 c.g.s.u. (Table III), a value more representative of inherently dissymmetric chromophores than of symmetric chromophores perturbed by a dissymmetric environment (41). Thus the asymmetric binding sites on or within protein molecules appear to be particularly effective perturbers of a symmetric chromophore. It is not surprising that the optical activity of aromatic chromophores or chromophoric cofactors incorporated into proteins is far greater than when the same chromophore is located in a small molecule and perturbed by a single asymmetric center. The aromatic chromophores of a phenyl group attached to a sulfonamide bound to carbonic anhydrase (30) show

3 Large crystals of the complexes of human isozyme B with both acetazolamide and azosulfonamide I have been prepared.

much larger induced rotatory power than the aromatic transitions of tyrosine or tryptophan (42, 43). The evident en- hancement of the optical activity of the aromatic transitions of the latter amino acids when incorporated into certain prot,ein environments including carbonic anhydrase has been com- mented upon previously (7, 27, 40, 44).

Positions and intensities of the azosulfonamide transitions are also altered by the protein environment. Changes in molecular environment which are known to perturb the transitions of the azo dyes can thus be used to construct some tentative hypotheses as to the nat.ure of the protein binding site. Transitions of the phenylazonaphthols are known to be sensitive to the polarity of the solvent (28, 45, 46). In the case of Compound I the less polar solvents produce hypochromic-bathochromic shifts of the principal absorption bands similar to those induced by the enzyme (Figs. 2 and 4). In fact, by adjusting a dioxane- water mixture from 10% dioxane to 96% dioxane, the contours and positions of the main bands can be made to correspond closely to the spectra of the several isozyme complexes. A hydrophobic cavity has recently been postulated to be present at the active center of carbonic anhydrase on the basis of infra- red studies of the competitive binding of carbon dioxide, azide, and nitrous oxide (47).

While a binding site composed of non-polar residues with ex- clusion of most water (with some variation between isozymes) would seem to satisfactorily account for the spectral shifts observed for the enzyme-bound azosulfonamide, several other factors must be considered. The formation of the conjugate acids of the phenylazo compounds and the consequent spectral shifts have been extensively studied in aqueous mineral acids (28, 46). While these acids normally have pK, values of -3 or -4 referred to the Ho scale (28, 48), it is conceivable that a hydrogen-bonded structure approaching the form of the acid compound could be formed with a group on the enzyme. The spectrum of Compound I in 50 y0 aqueous sulfuric acid, however, does not look like that observed for the enzyme-bound form of the azosulfonamide (Figs. 2 and 4).

Another hydrogen ion equilibrium to be considered is the dissociation of the proton from the sulfonamide group to form

--SOzNH + H+

/ --SOgNHz (IV)

\ -S02NH-Zn + H+

(V)

the anionic species (IV). This pKa varies from pH 6.6 to 10.4 for the sulfonamide inhibitors of carbonic anhydrase (10). In- direct evidence from studies on the pH dependence of enzymatic inhibition by these agents (49) and the pH dependence of the binding of a tritiated sulfonamide (6, 24) suggests that the anionic form (IV) is the inhibitory species. This fact coupled with the evidence that the sulfonamide group adds to the coordination sphere of the metal ion (7, 9, 30) suggests (V) as the bound form of the sulfonamide group. Titration data show Compound I to have a pK, at pH 10.6 which can be assigned to the sulfonamide group. Near pH 10 the major absorption band does undergo a hypochromic shift and some broadening (Figs. 2 and 4). The band position, however, does not shift appreciably. In addition to a non-polar environment,, the induction of the


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anionic form of the sulfonamide by metal ion binding must be considered as a possible factor contributing to the spectral shifts observed for the enzyme-bound form of Compound I. Chen and Kernohan (33) have recently reported their interesting study on the fluorescence of the complex formed between bovine carbonic anhydrase and 5-dimethylaminonaphthalene-l-sulfonamide. On the basis of the high quantum yield and the blue shift in the fluorescence spectrum of the bound sulfonamide, they also postulate a hydrophobic binding site and the dissociation of a proton from the sulfonamide group in the bound form. As has been suggested (6, 10) the sulfonamides must gain their high binding affinity from a cooperative or “chelate” effect involving both the ring binding forces and the interaction with the metal ion through the sulfonamide group.

The nature of the microenvironment producing the dissym- metry in the transitions of the bound form of the inhibitor is more complex. Stryer and Blout (50) have shown that binding of multiple dye molecules (including an azo dye) to polyglutamic acid induces large multiple Cotton effects at the wave lengths of the dye absorption bands. These Cotton effects are induced only by the helical forms of the synthetic polymer, disappear in the dye complexes of the random form, and are specific for the screw sense of the polymer helix. The Cotton effects for the a-helical poly-L-glutamic acid complex are mirror images of those for the a-helical poly-n-glutamic acid complex. Stryer and Blout differentiate “configurationally induced” optical rotatory power arising from the interaction of the symmetric dye chromophore with the local environment of the asymmetric a-carbon from “conformationally induced” optical rotatory power which might result from the interaction among several symmetric dye chromophores which have been ordered by the underlying polymer helix. The authors favor the latter mechanism to explain the induced optical activity in the case of the polyglutamic acid-dye complexes. In the case of carbonic anhydrase the conformationally induced rotatory power as a propert’y of a set of oriented dye molecules is not likely since only one azosulfonamide dye molecule is incorporated per molecule of carbonic anhydrase. Hence the non-zero product for pimicos0 induced for each transition must result from coupling with transitions of the protein (peptide or side chain transitions) or from dissymmetric electrostatic perturbations produced by charged groups or dipoles of the protein.

A comparison of the gaussian resolution of the ellipticity bands of the azosulfonamide complexes of two carbonic anhydrases, bovine B and human B (Figs. 9 and lo), reveals that both sign and magnitude of the induced visible ellipticity bands appear much more sensitive to local features of the stereochemistry surrounding the active center than to any basic underlying feature of the protein stereochemistry. These local stereo- chemical features must control the spatial relationships between the various geometric axes of the dye molecule and the perturb- ing groups on the protein. Such groups could be considered to produce a dissymmetric potential field around the symmetric chromophore as in the model suggested by Schellman (51). The resulting induced ellipticity would be especially sensitive to small changes in the surrounding protein structure or charge arising from amino acid substitutions if the sulfonamide is almost entirely surrounded by the protein as seems likely from t’he X-ray data (9). In addition, the geometry of the azosulfonamide is not completely fixed and deformations resulting from twisting about the essential single bonds 1-a and l’-LY’ (see Table

I, Compound II for nomenclature) are known to have major effects on the absorption spectrum (References 45 and 46; Fig. 13). The presence of large deep ultraviolet bands making significant contributions to the visible ORD of the complexes (Figs. 11 and 12) prevent any positive statements about possible conformational changes in the underlying protein structure induced by the sulfonamides.

On the other hand, a change in the nature of the metal atom at the active site, even from one active divalent metal ion, Zn(II), to another, Co(II), is sufficient to alter the rotatory power of the visible bands of the complex significantly (Fig. 8). Perhaps this is mediated through changes in the binding affinity of the sulfonamide secondary to changes in the coordination chemistry or to slight conformational changes in the active center. It has previously been demonstrated that inactive metal ions like Hg(II), even though bound at the active site of carbonic anhydrase, do not induce the binding of the azosulfonamide which leads t,o these induced ellipticity bands (8). The difference spectrum between the Co(I1) and Zn(I1) enzyme complexes (Fig. 8) does indicate that the mechanism of binding of the azosulfonamide, as far as its influence on the coordination sphere of the Co(I1) ion is concerned, must be very similar to the mechanism for the binding of acetazolamide (Diamox). The spectral shifts involving t,he d-d transitions of the Co(I1) ion induced by compound I are almost identical with those produced by acetazolamide (5, 7, 30). An additional feature supporting this conclusion is t’he negative ellipticity band appearing at 598 rnp in the Co(I1) enzyme-azosulfonamide complex. This corresponds to a negative ellipticity band observed previously for the long wave length d-d band of a number of Co(I1) carbonic anhydrase-sulfonamide complexes (30).

Any certainty about the assignments of the multiple ellipticity bands revealed by the gaussian analysis of the CD spectra to known transitions of the azobenzenes or azonaphthols is be- yond the scope of the present analysis. Information on precise band structure, however, can be gained from the simultaneous analysis of the absorption, CD, and ORD spectra. Close exam- ination of the absorption spectrum of Compound I in both bound (Fig. 2) and unbound state (Fig. 4) indicate that the large visible band is made up of at least two bands of comparable oscillator strengths. Yet the CD and ORD analyses (Figs. 9 to 12) indicate that one of these bands appears to gain the major rotatory power. Since single red crystals of these complexes containing 1 mole of azosulfonamide per mole of protein can be grown readily, it may be possible to do polarized absorption spectra and circular dichroism on single crystals of these highly colored enzyme-inhibitor complexes and relate the directional properties of both the absorption and ellipticity to the sulfonamide structure and to the molecular orientation of the protein. X-ray diffraction studies of the structure of some of the sulfonamide complexes now in progress (9) will likely provide additional information on the nature of the sulfonamide binding to carbonic anhydrase. Attempts to covalently couple some of the diazo- tized sulfonamides to surrounding groups on the protein, now in progress, may identify some of the primary protein structure in the active centers of these isozymes.

Acknowledgments-It is a pleasure to acknowledge the many helpful discussions and suggestions of Dr. Yash P. Myer of the State University of n’ew York, Albany, and his kindness in making available the circular dichroism instrument, the curve


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fitter, and the computer program for evaluating the Kronig-

Kramers transform. The excellent technical assistance of Mrs. Barbara Johnson is much appreciated.

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Joseph E. ColemanCarbonic Anhydrase-Azosulfonamide Complexes: SPECTRAL PROPERTIES

1968, 243:4574-4587.J. Biol. Chem.

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