Anion complexes of Cu(II) bovine carbonic anhydrase

ARCHIVES

OF

BIOCHEMISTRY

Anion LAURA

Institute

AND

Complexes

MORPURGO,’

ofBiological

BIOPHYSICS

Chemistry

170,

360-367

(1975)

of Cu(ll) Bovine

Carbonic

Anhydrase

GIUSEPPE ROTILIO, ALESSANDRO AND BRUNO MONDOVI and Institute of Applied Biochemistry, for Molecular Biology, Rome, Italy Received

December

University

FINAZZI

of Rome;

AGRG,

C.N.R.

Centre

18, 1974

1. The stability constants of some anion complexes of bovine Cu(I1) carbonic anhydrase have been determined. They are found to be three-four orders of magnitude higher than those of the corresponding complexes of bovine superoxide dismutase, aqueous copper, and copper compounds of low molecular weight. Stabilization can be achieved either through the additional formation of a hydrogen bond between the anion and the protein in some cases or through donor-acceptor or charge-transfer type interactions in halide complexes. 2. Room temperature experiments show that the EPR spectrum of the cyanide complex is unaffected by pH, so that the change observed by Taylor and Coleman (1) must be produced by freezing. 3. The properties-of halide complexes of Cu(II), Co(H), and Zn(I1) carbonic anhydrases are discussed and it is proposed that they give rise to pentacoordinate metal species. This may explain previous NMR data (2) without excluding water as a metal ligand at low pH. 4. The pK. = 8.0 and the HCOSbinding constant Wsco. = 3.5 x lo31 for Cu(II)BCA are discussed in relation to the lack of activity of Cu(I1) carbonic anhydrases.

The zinc ion contained in the carbonic anhydrases can be substituted by divalent cobalt without significant alterations of enzymatic activity (3) and of the active site geometry (4). The situation is completely different when divalent copper is substituted for the zinc ion, as the protein loses its activity (3) and the symmetry of the metal site changes from distorted tetrahedral to axial (1). However the Cu(I1) carbonic anhydrases retain many properties of the native enzymes: Cu(I1) is located approximately in the same site as Zn(I1) [within kO.1 A from X-ray data on human carbonic anhydrase C at 2 A resolution (4)]; it displays a comparable high affinity for monovalent anions (1); proton relaxation rate measurements show that Cu(I1) human carbonic anhydrase B coordinates a water molecule (5) like the cobalt enzyme (6). In view of these considerations we have tried to investigate in more detail the prop1 On leave iia de1 C.N.R.

from

the Laboratorio

erties of Cu(I1) bovine carbonic anhydrase in the attempt to understand the reasons for the complete absence of activity in this enzyme in spite of the fact that the metal appears to be coordinated to the same hystidy1 residues (7) and to a water molecule (5) as in the native enzyme and to display a comparable chemical reactivity. We have reexamined the chemical and physical properties of the anionic derivatives of Cu(I1) bovine carbonic anhydrase, as the anions have proved in so many instances to be a sensitive probe of the active site of metalloenzymes (8). MATERIALS

0 1975 by Academic Press, of reproduction in any form

di Cromatogra-

Inc. reserved.

METHODS

Bovine carbonic anhydrase (BCA) was obtained as a, by-product in the preparation of superoxide dismutase according to McCord and Fridovich (9). The first peak eluted from the DEAE cellulose column has carbonic anhydrase activity. The fractions collected at the very top of the peak gave a single band on starch gel electrophoresis and were used without further purification, after concentration by ultradialysis. The removal of zinc was achieved by incubating the protein in the presence of o-phenan360

Copyright All rights

AND

ANION

COMPLEXES

OF

Cu(I1)

BOVINE

CARBONIC

361

ANHYDRASE

lected the positions and intensities of extra bands, generally very intense, which appear upon reaction with anions. Two new bands are formed by reaction with II. With Br- the solution turns yellow but only a very ill-defined shoulder can be detected around 400 nm together with a general increase of the near uv absorption. A single band, or none at all, is observed with the other anions. EPR spectra. In Fig. 2 are reported the X-band EPR spectra, measured at - 17O”C, of Cu(II)BCA and of its Cl-, Br-, and Iderivatives. An increasing degree of rhombicity is observed in the series. The spec-

throline according to Lindskog and Malmstrijm (101. Copper carbonic anhydrase was prepared by addition of slightly less than stoichiometric amounts of standardized 0.1 M copper sulphate solutions. The esterase activity of the protein was assayed by the method of Packer and Stone (11). Absorption spectra were obtained with a Beckman DK-2 spectrophotometer, equipped with a water circulating cell-holder in the equilibrium constants experiments. The temperature was of 22 f

0.5”C. Electron paramagnetic resonance spectra were recorded with a Varian V-4502-14 spectrometer. B3Cu (>96%) was obtained from Oak Ridge National Laboratory in the form of CuO, which was dissolved in H,SO,. RESULTS

Optical spectra. In the first column of Table I are collected the data relative to the d-d transitions of the optical absorption spectrum of the copper chromophore of Cu(II)BCA and of its anionic derivatives. Some spectra are reported in Fig. 1. The broad asymmetrical band at 770 nm in the native enzyme is generally shifted by the anions toward higher energy, the more the stronger the anion in the spectrochemical series. A small shift in the opposite direction is produced by halides. With the exception of HC03- and Cl-, anions cause an increase of the molar extinction coefficient. In the second column of Table I are col-

600

d-d(c)

I

Bovine

carbonic

anhydrase

W’)

Kd

~-‘)a

Kz.( M-‘)c

-

1.1

x 103

pK 9.0

Hz0

770(130)

I-

790(400)

445(2800) 355(3200)

1.9

x 104

BrCl-

790(300)

400(sh) -

7.0

x 102

1.2

x lo2

790(130)

1.7

x 102

5.0 4.0

x 10 x 10

CNN3CNSCNOHCOIW’Oa

690(190) 735(330)

400(2700)

735(150) 735(160)

360(900) -

a From b From r From

ref. 14. ref. 13. ref. 22.

Superoxide

diamutase,

C.T.(r) K,“(

735(140)

1( 10

FIG. 1. d-d Absorption bands of Cu(II)BCA and of its anion complexes. (a) 1.3 mM unbuffered aqueous enzyme; (b) plus 3.0 mM CN-; (c) plus 2.0 mM N3-; (d) plus 20 mM II. The pH of the solutions was about 6. The temperature was 25°C.

TABLE

A&Xl

600 wovelengthlnml

-

pK=8

-108 1.1 x 106 4.6 x -3 x 3.5 x 6.7 x

lo5 105 103 lo3

= 7 x 102

x 10 x 10 5.0 8.0 x lo6

d-d(c) 680(120)

C.T.(e) -

350(sh)

K,.( M-‘)
8.0 2.5

lo4 104 1.3 x lo5 2.0 x 102

555(110) 670(120) 670(120) 670(80)

37X1200) 350(800) -

1.5 x 102 (102
362

FIG. 2. EPR spectra of “Cu(II)BCA and of its halide complexes at -170°C. (a) 2 mM unbuffered aqueous enzyme; (b) plus 0.5 M NaCl; (c) plus 0.2 M KBr; (d) plus 0.02 M KI. Microwave frequency 9.15 GHz. Microwave power 20 mW. Modulation amplitude 10 gauss.

trum of the iodide derivative was measured in a number of different conditions, changing I- concentrations (0.1-0.02 M), temperature (the room temperature spectrum is reported in Fig. 4) and using @X!u, with almost identical results. The spectra of other anion derivatives are very similar to those measured by Taylor and Coleman (1) for Cu(I1) human carbonic anhydrase B. All of them, except the high pH CNand HC03-, show a distinct rhombic line shape. The low temperature spectrum of the CN- complex is strongly pH dependent (Fig. 3) as reported for Cu(I1) human carbonic anhydrase B (1). Such pH dependence however can not be observed at room temperature (Fig. 4b). In this casethe spectrum is insensitive of pH up to pH 10 as it is the optical spectrum in the same pH r region. In Fig. 4 are also reported the room temperature EPR spectra of other anionic derivatives. Binding constants of anionic derivatives. In Fig. 5 is reported a typical titration of Cu(II)BCA with II. From the increase of absorption at 450 run the ratio [EHZl/([Tl - [EHII) can be calculated. [Tl is total enzyme concentration. When this ratio is plotted vs free I- concentration a straight line through the origin is obtained, indicating 1:l ratio of protein to ligand. The slope of the line should correspond to the equilibrium constant of the reaction

EH+ + I-

* EHI

K , = [EHIl/[EH+l[I-1,

(1)

where EH+ indicates a form of the enzyme which is predominant at low pH. However different slopes are obtained by changing pH, or buffer concentration or buffer anion. In Fig. 6 are reported as an example the plots relative to several concentrations of carbonate buffer pH 9.9. Analogous effects on anion binding constants had already been observed by Packer and Stone (12) on native bovine carbonic anhydrase from inhibition experiments. They can be rationalized by also considering the following equilibria: EH+ + B- G EHB KB = [EHBlI[EH+l[B-I EHf%E

(2)

+H+ K, = [E1[H+l/[EHfl,

(3)

where B- is the concentration of the buffer monovalent anion and E is the form of the enzyme predominant at high pH. From a formal point of view the equation EH+ + OH- % E*H,O is equivalent to Eq. (3) which at present we use for the sake of simplicity. Further comments on this formulation will be reported later on. By combining the equations for the three equilibrium constants with the conservation equation

ANION

[Tl = L!mIl

COMPLEXES

OF

+ [Em31 + [EH+] + [El

Cu(II)

BOVINE

(4)

it is obtained: -1

=- 1

K BPP KI

(5) K,

+ KW+l

+ 5 [B-l, K,

where K,,, is the slope of each line of Fig. 6. In Fig. 7A the reciprocal of K,,, is plotted vs [H,PO,-1, that is the concentration of the buffer anion B- binding to the enzyme, calculated assuming that 30% of the buffer is in this form at pH 6.4. Figure 7B shows the analogous plot relative to carbonate buffer pH 9.9, where the concentration of HC03- has been taken to be 50% of total buffer concentration. In Table I, third column, are reported the values of equilibrium constants obtained from intercept and slope of the lines of Fig. 7A and 7B. The consistency of the calculated values for K, and KI was checked by plotting the absorbance at 450 nm of a buffer-free solu-

2600

2800 Magnetic

ANHYDRASE

363

tion of the enzyme in the presence of KZ vs pH (Fig. 8). The solid lines represent calculated titration curves, using K values of Table I (lower line) and changing the value of fla to 8.1 (upper line) to get a better fit. The difference is small and not relevant to the following discussion. In the case of Ns- and CNS which react smichiometrically at low pH the stability constants were measured at pH 9.9, in carbonate buffer. In this case Eq. (5) can be applied in the form KNJKaPPJ = KHCOI [HCO,-] as the intercept on the ordinate axis is close to zero, due to the high values of the binding constant. The values for CN- were obtained by competition with NB- in carbonate buffer pH 9.9, Br- and Cl- constants were evaluated by competition with I- in low pH unbuffered solutions as these anions do not give rise to very intense bands. DISCUSSION

Cu(II)BCA shows a surprisingly high affinity for monovalent anions as previously

3000 tleld

CARBONIC

3200

~gouss)

FIG. 3. EPR spectra of Cu(II)BCA and of its CN- complexes at -170°C. (a) 1.2 mM unbuffered aqueous enzyme; (b) 1.2 mM KCN; (c) 10 mM KCN and 10 mM carbonate buffer pH 9.9. Microwave frequency 9.15 GHz. Microwave power 20 mW. Modulation amplitude 10 gauss.

364

MGRPURGC

ET

AL.

ionic derivatives in the native enzyme as well as in the copper substituted enzymes. This type of bonding should be particularly fit to HC03- which is supposed (16) to be the substrate in the dehydration reaction, and whose structure is similar to that of the sulfonamide group (17). The EPR spectrum of the HC03- bound Cu(II) enzyme is axial and the d-d band has relatively low intensity. When other anions of different size and shape such as N3-, CN-, CNO-, etc., are bound in place of HCO,- a

I

v I 2600

field

I

i n

I --

I -.--

--

I

I 3600

I 3100 Magnetic

1.0

igauss)

FIG. 4. Room temperature EPR spectra of Cu(II)BCA and of some of its anion complexes. (a) 1.2 mM unbuffered aqueous enzyme; (b) 10 mM KCN and 10 mM carbonate buffer pH 9.9; (c) 1.2 mM NaN,; (d) 1.2 mM MeSH; (e) 0.12 M KI. Microwave frequency 9.5 GHz, other parameters as in Fig. 3.

found with the native (12) and cobalt substituted (13) carbonic anhydrases. A binding constant for Ns- of 1.2 x lo6 M-’ compares with values as low as 1.5 X 10’ M-’ for the analogous derivatives of superoxide dismutase (141, Cu(I1) diethylenetriamine (141, and for aqueous copper (15). An interaction of the anions with other protein groups (13) besides the metal ion is thought to be responsible for this high affinity and the possibility was suggested (12) of ion-pair formation between anions and an imidazolium group. From the ORTEP stereodrawings (4) of the residues surrounding the zinc ion in human carbonic anhydrase C it appears that threonine 197 could be hvdrogen bonded to the water molecule at the &c site. In the presence of 3-acetoxymercuri-4aminobenzene-l-sulfonamide, a powerful inhibitor of carbonic anhydrases, the same threonine 197 residue appears to be hydrogen bonded to one atom of the sulfonamide group bound to zinc (4). Analogous hydrogen bonds from threonine 197 could account for the increased stability of the an-

al c”

0.5 _-._.-_.

!

..‘

‘-i,r.,,‘I (I/,, \ .( ,’ 1 ‘.(

!

0

I’ 300

I 400

500

Wavelength FIG. 5. The reaction of Cu(II)BCA with KI. 0.38 mM enzyme, 29 mM carbonate buffer pH 9.9; KI: 6.5; 12.8; 19.3; 25.6; 31.8; 38.0; 50.5; 62.6; 120 mxr.

ANION

0

2

COMPLEXES

4 [I‘]

6

OF

Cu(II)

6

BOVINE

10

lo2

FIG. 6. The reaction of Cu(II)BCA with KI. Enzyme concentration 0.35 mM, carbonate buffer pH 9.9: (a) 0.029 M; (b) 0.057~; (c) 0.10 M; (d) 0.18 d.

FIG. 7. The dependence of K.,, of Cu(II)BCA-Icomplex on buffer concentration. A: phosphate; carbonate.

B:

rhombic component (1) is found in the EPR spectrum and the intensity of the d-d bands increases. The metal site is clearly distorted. This appears to be an inverse process to that observed in superoxide dismutase where anion binding causes a change from rhombic to axial symmetry of the copper site (14). Taylor and Coleman (1) also report the cyanide spectrum to be axial at pH 8, while a rhombic line shape is apparent at pH 6. Our observations at room temperature indicate that the CNcomplex retains its rhombicity at pH 10 so that the change to axial symmetry must be due to freezing besides pH. Also the optical spectrum does not change in this pH range. The shift of the d-d bands to shorter wavelengths caused by cyanide is considerably smaller in Cu(II)BCA than in superoxide dismutase (14), as if the anion were prevented from a close interaction with the metal. It is possible that a larger shift occurs in the axial form of the deriva-

CARBONIC

ANHYDRASE

365

tive, so similar to that of the CN- treated superoxide dismutase. Such a behaviour is never observed with other anions which do not form with Cu(I1) complexes of high stability, as CN- does at sufficiently high pH (18), in the absence of additional factors. Thus it appears that the stability of all these complexes depends in a critical way on a number of factors as the physicochemical properties of the anion, the pH, and the protein conformation which can be in some way altered by freezing. In Co(II) carbonic anhydrases freezing causes a change from high to low spin Co(B) in the presence of excess CN- (19, 20). Also in this case the anion appears to be brought closer to the metal upon freezing. The above picture, involving formation of a hydrogen bond to explain the high stability of carbonic anhydrases anion complexes does not hold for halide complexes as their stability increases from fluoride to iodide (see Table I and references therein), while the inverse order would be expected on the basis of either hydrogen bonding or metal ion complexing ability. Their behaviour is also peculiar in other respects: (i) the d-d band of Cu(II)BCA is shifted to slightly lower energy by halides unlike other anions; (ii) the d-d bands of Co(II)BCA halide complexes have extinc-

FIG. 8. pH dependence of the reaction of Cu(II)BCA with KI. 0.17 mM enzyme, 20 mM KI. Solid lower line: calculated for pE. = 8.0 and K, = 1.9 x lo4 I+-‘; solid upper line: calculated for pE, = 8.1 and KI = 1.9 x 10’ M-I.

366

MORPURGO

tion coefficients (16) lower than most of the other species; (iii) the metal-halide distance in Zn(I1) human carbonic anhydrase C are greater than generally found in small complexes (21); (iv) the logarithm of the stability constant of Zn(I1) (22), Co(II) (13), and Cu(II)BCA halide complexes depends linearly (Fig. 9) on the redox potential of the XdX- couple (23), a type of correlation generally found in chargetransfer or donor -acceptor complexes (24)) within a series of related compounds; (v) relaxation data for solvent water protons (6) and 35C1- ions (25), in solutions of Co(H) and Zn(II) carbonic anhydrase, respectively, indicate that, at low pH, the binding Cl- cannot be competing with a water molecule for the metal site (21). The latter point was taken to imply that there is no water at the metal site and that Cl- is competing with a residue on the protein (21). By considering all of the points above, we would rather suggest that halides enter as a fifth ligand into the metal coordination sphere bringing about a change of coordination number from four to five. The d-d spectra of both Cu(II) and Co(I1) enzymes are consistent with this hypothesis. Per&coordinate high-spin Co(I1) complexes seem to be characterized (26) by absorption coefficients of d-d transitions lower than 250 what is found for Cobalt carbonic anhydrase. The optical spectra of Cl-, Br-, and I- derivatives of Cu(II)BCA are closely similar to the corresponding derivatives of bisdipyridyl Cu(II) (27), which have a compressed trigonal bipyramidal structure (28). However the geometry of Cu(II)BCA halide complexes appears different to some extent. Their EPR spectra are axial in the Cl- derivative and increasingly rhombic in the Br- and I- derivatives and could rather indicate a distorted square pyramidal structure. Of course the difference between trigonal bipyramidal and square pyramidal stereochemistry is relatively small and a clear-cut distinction is difficult (29). If a different coordination to carbonic anhydrases is assumed for halides and the anions previously discussed, also the mechanism of stabilization may be different. Point (iv) suggests that the halide com-

ET AL.

I 0

1

I 2

I 3

I 4

I 5

log K FIG. 9. Relationship between the stability conatant of Zn(I1) (A--A), Co(I1) (O--O), and Cu(II)BCA (O--O) complexes with halides and the redox potential of XJX- couples.

plexes are stabilized by donor-acceptor interactions. This is supported by the presence of intense charge-transfer bands in the iodide derivative of Cu(II)BCA, and, at higher energy, in the bromide. Only a weak shoulder at about 350 nm is observed by reacting superoxide dismutase with 0.5 M KI (Morpurgo, L., unpublished results) while the azide or thiocyanate charge transfer bands are only shifted by 25 and 10 nm, respectively, toward lower wavelengths in superoxide dismutase (14) with respect to Cu(II)BCA. This type of interaction could explain the large Zn-halide distance (21) measured by X-rays in Zn(II) human carbonic anhydrase, which are comparable to the distance observed in the Iand Cl- donor-acceptor complexes of nicotinamide (30). This could mean that the halide interacts not only with the metal ion but again with some closely located protein residue. This picture of the reaction of halides with carbonic anhydrases can explain NMR data without conflicting with the widespread belief (16, 31) that H,O is a ligand of the low pH form of carbonic anhydrase and that this is the group ionizing at pH 7-8 in the various isozymes (32). The experimental finding that the fla of this group and the stability of the halide complex are interdependent is explained by the fact that both processes tend to put a negative charge on the metal site, which has low affinity for divalent anions (13). It is interesting to note that the binding of

ANION

COMPLEXES

OF

Cu(I1)

carbon dioxide to low molecular weight complexes is often dependent upon the presence of a hydroxyl group (31,33) bound to the metal. MX(CO)(Ph,P), complexes (33) (M = Rh, Ir; Ph,O = triphenylphosphine) do not absorb COZ when X = F-, Cl-, C104-, but they do when X = OH-. Moreover the CO* adducts are readily converted into bicarbonate derivatives in ethanol (33). This fact suggests that the -OH group of Threonine-197 may have a role in the catalytic mechanism, that is by hydrogen bonding the COZ molecule it could facilitate its polarization and attack by the OH-, as shown in the following scheme : zn

0

/H 4

I

/

,H lhri1971

/H

zn 0 \

C-L

HThrl1971

BOVINE

8. MORPURGO,

9. 10. 11.

12. 13. 14. 15.

16. 17. 18.

ACKNOWLEDGMENTS

24.

4.

5. 6.

7.

COLEMAN,

J. E. (1971)5.

Biol.

246, 7058-7067.

R. D. III (1972) Proc. Nat. Acad. Sci. USA 60, 2422-2425. LINDSKOG, S. AND NYMAN, P. 0. (1964)Biochim. Biophys. Acta 85, 462-474. LINDSKOG, S., HENDERSON, L. E., KANNAN, K. K., LIWAS, A., NYMAN, P. O., AND STRAND. BERG, B. (1971) The Enzymes, Vol. 5, pp. 587665, Academic Press, New York. KOENIG, S. H. AND BROWN (III), R. D. (1973) Ann. N.Y. Acad. Sci. 222, 752-763. FABRY, M. E. (RIEPE), KOENIG, S. H., ANDSCHIL LINGER, W. E. (1970) J. Biol. Chem. 245, 4256-4262. TAYLOR, J. S. AND COLEMAN, J. E. (1973)J. Biol. Chem. 248, 749-755.

2. KOENIG, 3.

J. S. AND S. H. AND

BROWN,

22.

useful discusassistance.

REFERENCES Chem.

20. 21.

23.

We thank S. H. Koenig for much sion and P. Gerosa for able technical

1. TAYLOR,

19.

25. 26. 27. 28. 29. 30. 31. 32. 33.

ANHYDRASE

367

L., FINAZZI AGR~, A., ROTILIO, G., B. (1974) Biochim. Biophys. Acta 336, 324-328. MCCORD, J. M. AND FRIDOVICH, I. (1969) J. Biol. Chem. 244, 6049-6055. LINDSKOG, S. AND MALMSTR~M, B. G. (1962) J. Biol. Chem. 23’7, 1129-1137. POCKER, Y. AND STONE, J. T. (1967)Biochemistry 6, 668-678. POCKER, Y. AND STONE, J. T. (1968)Biochemistry 7, 2936-2945. LINDSKOG, S. (1966) Biochemistry 5, 2641-2646. MORPURGO, L., GIOVAGNOLI, C., ANDROTILIO, G. (1973)Biochim. Biophys. Acta 322,204-210. SILL~N, C. G. AND MARTELL, A. E. (1964) Stability constants of metal ion complexes. Chem. Sot. (London) Special Publication 17. LINDSKOG, S. AND COLEMAN, J. E. (1973) Proc. Nat. Acad. Sci. USA 70, 2505-2508. MATHEW, M. AND PALENIK, G. J. (1974) J.C.S. Perkin 11, 532-536. ROTILIO, G., MORPURGO, L., GIOVAGNOLI, C., CALABRESE, L., AND MONDOV~, B. (1972) Biochemistry 11, 2187-2192. HAFFNER, P. H. AND COLEMAN, J. E. (1973) J. Biol. Chem. 248, 6630-6636. COCKLE, S. A. (1974) Biochem. J. 137, 587-596. BERGST~N, P. C., WAARA, I., L~VGREN, S., LIG JAS, A., KANNAN, K. K., AND BENGTSSON, U. (1972) in Oxygen affinity of Hemoglobin and Red Cells Acid Base Status (Rerth, M. and Astrup, P. eds.), pp. 363-383. Muuksgaard, Copenhagen and Academic Press, New York and London. LINDSKOG, S. (1970) Struct. Bonding (Berlin) 8, 153-196. Handbook of Chemistry and Physics (1968-1969) 49th Edition (R. C. West, ed.), p. D-86. The Chemical Rubber Co. FOSTER, R. (1969) Organic Charge Transfer Complexes, Academic Press, London and New York, p. 190. WARD, R. L. (1969)Biochemistry 8,1879-1883. ROSENBERG, R. C., ROOT, C. A., AND GRAY, H. B. (1975) J. Amer. Chem. Sot. 97, 21-26. ELLIOTT, H., HATAWAY, B. J., AND SLADE, R. C. (1966) J. Chem. Sot. (A), 1443-1445. BARCLAY, G. A., HOSKINS, B. F., AND KENNARD, C. H. L. (1963) J. Chem. Sot., 5691-5699. FURLANI, C. (1968) Coord, Chem. Rev. 3, 141167. FREEMAN, G. R. AND BUGG, C. E. (1974) Acta Cryst. B 30, 431-443. CHAFFEE, E., DASGUPTA, T. P., AND HARRIS, G. M. (1973) J. Amer. Chem. Sot. 95,41694173. WARD, R. L. (1970)Biochemistr-y 9,2447-2454. FLYNN, B. R. ANDVASKA, L. (1974)J.C.S. Chem. Comm., 703-704. AND

’

In the kinetic mechanism of Lindskog and Coleman (16) the following step is the dissociation of HC03-. Respect to the native enzyme, Cu(II)BCA has a higher pK,, comparable to that of the less active human carbonic anhydrase B (321, and a higher HCOB- binding constant. Both are unfavourable factors in the kinetic mechanism and can account to some extent for the lack of activity of the copper enzyme.

CARBONIC

MONDOV~,