A spectropolarimetric investigation of bovine cobalt carbonic anhydrase

534

SHORT t ;OM ?vlU N ICATI ( ) N ~.

Canada thistle and tomato, and in r~ots, stems, and leaves of light-grown bean plants. The presence of this enzvlne and of OMP pyrophosphorylase 2,'~ sut)ports the previous conclusions based on inhibitor and labeled metabolite studiesV, s that the, normal pathway for UMP formation in plants proceeds via orotic acid and OMP. This work was supported bv Grant GB-9o 7 from the National Science Foundation. The helpful technical assistance of Miss A. JOHNSON was appreciated.

Department of Botany and Plant Pathology, Colorado State Universi(v, Fort Collins, Colo. (U.S.A.) I 2 3 4 5 6 7 8 9

,JOHN H . W O L C O T T

CL~,:oN Ross

I. LIEBERMAN, A. KORNBERG, AND E. SIMMS, J. Biol. Chem., 215 (I955) 403. M. KAPOOR AND E. WAYGOOD, Canad. J. Biochem., 43 (1965) 143. J. \¥OLCOTT AND C. ROSS, Plant Physwl., 4 ° (z965) (Suppl.) lxii. W. CREASEY AND R. HANDSCHUMACHER, dr. Biol. Chem., 236 (1961) 2058. D. BLAIR AND V. POTTER, dr. Biol. Chem., 236 (I961) 2503 . D. KASBEKAR, A. NAGABHUSHANAM AND D. GREENBERG, J. Biol. Chem., 239 (1904) 4245. C. R o s s , Biochim. Biophys. Acta, 87 (1964) 564 . C. R o s s , Plant Physiol., 4 ° (1965) 65. J- B u c H o w l c z AND I. I~-EIFER, Acta Biochim. Polon., 8 (1961) 25.

Received April I8th, 1966 Biochim. Biophvs. Acta, 122 (1966) 532-534

BBA 63197

A spectropolarimetric investigation of bovine cobalt carbonic anhydrase Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) is a colorless protein containing one zinc ion per molecule'. A colored, enzymically active carbonic anhydrase containing cobalt at the metal-binding site can, however, be made 2. The visible absorption spectrum of the cobalt enzyme is sensitive to changes in the environment of the active site. This property has been utilized to obtain information about the interaction of the enzyme with protons and inhibitors 3. Studies of various physical properties of the cobalt enzyme might be expected to yield information about the structure of the chelating site. Thus, the similarities between the visible absorption spectra of the cobalt enzyme and tetrahedral Co(II)-complexes have been pointed out3, a. The present communication deals with the optical rotatory dispersion (ORD) of cobalt carbonic anhydrase. In the visible range, small but significant deviations from the simple dispersion curves of the zinc enzyme and the apoenzyme were found. The binding of anionic inhibitors has only relatively small effects on the rotatory pattern. In the complexes with sulfonamide inhibitors, however, a considerable enhancement of the optical activity of the visible absorption band was observed. Removal of zinc from carbonic anhydrase and reactivation with cobalt do not affect the Cotton effectsS, 6 present in the 25o-3oo-m# range. A b b r e v i a t i o n : O R D , optical r o t a t o r y dispersion.

Biochim. Biophys. Acta, 122 (1966) 534-537

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Fig. i. U l t r a v i o l e t O R D of b o v i n e carbonic a n h y d r a s e . [a] = specific rotation. T e m p e r a t u r e , 25°; 0.025 M i m i d a z o l e - H C l buffer, (pH 6.5); p r o t e i n concn., a p p r o x . I m g / m l . ( ), zinc e n z y m e , a p o e n z y m e , or cobalt e n z y m e . E s t i m a t e d u n c e r t a i n t y in [a], 20 °. ( . . . . . ), a c e t a z o l a m i d e - i n h i b i t e d zinc e n z y m e or c o b a l t e n z y m e . T o t a l i n h i b i t o r concn., a p p r o x . 5 ' lO-5 M. (--- - - ) , zinc e n z y m e in 8 M urea. Insert: difference O R D ([a] for zinc e n z y m e plus a c e t a z o l a m i d e minus [a] for zinc e n z y m e ) . A c e t a z o l a m i d e has an a b s o r p t i o n m a x i m u m at 270 m # (e~ = 7900 M -1" cm-1). Fig. 2. Visible O R D and a b s o r p t i o n spectra of b o v i n e carbonic a n h y d r a s e . [al -- specific rotation. T e m p e r a t u r e , 25°; 0.025 M imidazole HC1 buffer, (pH 6.5) or 0.025 M t r i e t h a n o l a m i n e buffer, (pH 8.8); p r o t e i n concn., i i . T m g / m l . (--), zinc e n z y m e or a p o e n z y m e ; ( ), cobalt e n z y m e , p H 8 . 8 ; (. . . . . ), cobalt e n z y m e , p H 6 . 5 , w i t h p o t a s s i u m c y a n i d e (8-1o 4M); (. . . . . ), c o b a l t e n z y m e , p H 6.5, w i t h p o t a s s i u m c y a n a t e (8-1o -4 M); ( - - - - - - ) , cobalt e n z y m e , p H 8.8, w i t h a c e t a z o l a m i d e (8. lO -4 M).

Bovine carbonic anhydrase B was purified from red blood cells according to the m e t h o d of LINDSKOG 1. The preparation of a p o e n z y m e and cobalt e n z y m e have been described2, 3. E n z y m e concentrations were e s t i m a t e d spectrophotometrically using the eM value at 280 m # of 57 00o M - 1 . c m -1 and a molecular weight of 30 ooo (ref. 7). O R D m e a s u r e m e n t s were performed at 25 ° w i t h a Cary Model 60 recording spectropolarimeter using a I o - m m t h e r m o s t a t e d cell. Once the base line had been recorded, a set of m e a s u r e m e n t s was performed w i t h o u t r e m o v i n g the cell from its holder. To obtain full details of Cotton effects, portions of the curves were rerun slowly at a low noise level (time constant, i o sec), using the 0.02 ° full range setting. The zero suppression control was used to keep the curves on the chart paper. The O R D of zinc carbonic anhydrase above 31o mff follows a p p r o x i m a t e l y a o n e t e r m Drude equation w i t h [a]D -- - - 5 8 i 2 ° and ;re = 222 ± 2 mff, in close agreem e n t with preliminary results 2. As was also found earlier, the r e m o v a l of zinc does not change the O R D in this w a v e l e n g t h region to any detectable extent. In the 250 3oo-mff range, zinc carbonic anhydrase shows a number of Cotton effects which disappear on denaturation with urea (Fig. I). Neither the r e m o v a l of zinc, nor reactivation with cobalt, resulted in any measurable change in the pattern of these Biochim. Biophys. Acta, 122 (1966) 534-537

53()

SHORT C(),MMUNI(AT1ON5

Cotton effects. The visible absorption band of the cobalt enzyme (pH ,~.S) sh(~\v~ ~,nh a very weak optical activity (Iqg. 2). The binding of anionic inhibitors changes the ORD of the cobalt enzyme slightly as illustrated for (~N and N(70 in Vig. 2. Similar, but not identical, changes were ohtained when CI (~r NO:, were bound. Fig. 2 also shows the ORI) ()f the cobalt enzvme in the presence of the ~ulfonamide inhibitor, acetazolamide (2-acetylamino-I,3,4-thiadiazole-5-sulfonamide, l)iamox). Although the absorption spectra of the acetazolamide and, for example, the cyanate complexes art, quite similar, the ORD of the two are very different. A positive, asymmetric Cotton effect of considerable amplitude (molar rotation, M 4ooo ) was found for the acetazolamide-inhibited enzyme, but not for the eyanate-inhibited enzyme. Sulfanilamide gave a similar Cotton effect ([M 3ooo ). In the ultraviolet region, the acetazolamide-inhibited enzyme exhibits an additional (~otton effect superimposed on the protein Cotton effects (Fig. ~). Acetazolamide gives the same effect in the ultraviolet region with the zinc enzyme, but not with the apoenzyme, which has been shown not to bind this inhibitor :~. On the basis of the ORD of aromatic amino acid derivatives, ROSENBER(;s has suggested that the Cotton effects of bovine carbonic anhydrase in the 25o-3oo m# range are almost entirely clue to tryptophan. Tyrosine, as well as tryptophan, is probably contributing s to the similar, but more complex, Cotton effects found in human carbonic anhydrases B and C (refs. 5 and 6). (The nomenclature of these enzymes is found in ref. 7.) Apparently, at neutral pH, the metal ion is not essential for maintaining the asymmetric structure giving rise to these effects either in the bovine enzyme as shown here or as shown bv others 8,9, in the human enzymes B and C. The sensitivity of the absorption spectrum of the cobalt enzymes to changes in the solvent environment indicates that the metal ion is situated at the surface of the protein, perhaps in a "crevice". However, acid difference spectra indicate that all the tryptophan residues of the bovine e n z y m e (NILSSON AND LINI)SKO(L unpublished results) and the human enzymes (RIDD1FORI) 1°) a r e buried in the interior of the molecule. It thus seems likely that the asymmetric aromatic structure and the chelating site are in difl)rent parts of the enzyme molecule. The ORD patterns of the bovine cobalt enzyme and its inhibitor complexes are quite similar to those found by COLEMAN9 in an independent study on human carbonic anhydrase B. It seems probable that the metal ion coordination is identical in the two enzymes (see ref. 4), but that differences in the environment of the chelating sites give rise to minor differences in various physical properties. For example, the acetazolamide-inhibited enzymes both show distinct Cotton effects with peaks at closely the same wavelengths and with almost the same amplitudes. Apparently the introduction of a "bulky" sulfonamide molecule into the coordination sphere of the cobalt ion makes the complex more optically active than does the binding of a relatively small anion. For the human enzyme, however, no effect of acetazolamide was found in the ultraviolet range of the ORD curve 9. This may be due to a structural difference also reflected in the stronger interaction of this inhibitor with the bovine enzyme than with human enzyme B (P. J. WlSTRANI), personal communication). The detailed structures of the active sites of the two enzymes can, of course, not be identical, since the catalytic properties are different~, 7. Predictions about the symmetry of metal coordination are not feasible, unless the ORD data are combined with other physical properties of the complex. MeasureBiochim. Bioph3's. Acta, i22 (tqOt)) 53-t 537

537

SHORT COMMUNICATIONS

ments of near-infrared spectra and magnetic properties are in progress. The crystal structure of human carbonic anhydrase C is being investigated n. A three-dimensional Fourier synthesis at 5.5 A resolution has been obtained (B. STRANDBERG, personal communication), but at this resolution, much detail of the chelating site cannot be discerned. I wish to thank Dr. JULIAN M. STURTEVANT,in whose laboratory this work was carried out, for many valuable discussions. This investigation was financially supported by research grant GM-o4725, the enzyme preparations by research grant GM-I228o-o2, from the Institute of General Medical Sciences, U.S. Public Health Service.

Department of Chemistry, Yale University, New Haven, Conn. (U.S.A.) I

2 3 4 5 6 7 8 9 IO II

SVEN LINDSKOG*

S. LINDSKOG, Biochim. Biophys. Acta, 39 (196o) 218. S. LINDSKOG AND g. G. MALMSTR~M, J. Biol. Chem., 237 (1962) 1129. S. LINDSKOG, J. Biol. Chem., 238 (1963) 945. S. LINDSKOG AND P. O. NYMAN, Biochim. Biophys. Acta, 85 (1964) 462. A. ROSENBERG, Federation Proc., 24 (1965) 413 . D. V. MYERS AND J. T. EDSALL, Proc. Natl. Acad. Sci. U.S., 53 (1965) 169. p. O. 2qYMAN AND S. LINDSKOG, Biochim. Biophys. Acta, 85 (1964) 141. A. ROSENBERG, J. Biol. Chem., in the press. J. E. COLEMAN, Biochemistry, 4 (1965) 2644. L. M. RIDDIFORD, J. Biol. Chem., 240 (1965) 168. B. TILANDER, B. STRANDBERG AND K. FRIDBORG, J. Mol. Biol., 12 (1965) 74 o.

Received May i8th, 1966 * P r e s e n t address: Sweden.

D e p a r t m e n t of Biochemistry, University of G6teborg, G6teborg,

Biochim. Biophys. Aeta, 122 (1966) 534 537

BBA 63206

Kinetic properties and mechanism of action of human heart lactate dehydrogenase and m-hydroxybutyrate dehydrogenase After myocardial infarction or liver disease, both lactate dehydrogenase (Llactate:NAD oxidoreductase, EC 1.1.1.27) (LDH) and "a-hydroxybutyrate dehydrogenase" activities are elevated in the serum, but to different extents 1. This has led some authorsZ, 3 to postulate that lactate dehydrogenase and a-hydroxybutyrate dehydrogenase may be different enzymes, whereas other studies4, 5 have indicated that only one enzyme is involved. The present work shows that, in the case of LDH 1 (H4) of heart, both activities reside on the same enzyme. Lactate dehydrogenase was extracted from human heart muscle by homogenizing in 8 mM sodium phosphate buffer-I mM EDTA. The supernatant was concentrated by addition of ammonium sulphate to 80% saturation. The precipitate so formed was collected by centrifuging and dissolved in 50 mM buffer (pH 7.0). Abbreviation: L D H , lactate dehydrogenase.

Biochim. Biophys. Acta, 122 (1966) 537-54 °