Venom exonuclease

331

Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 3 3 1 - - 3 3 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38384

VENOM EXONUCLEASE II. AMINO ACID COMPOSITION AND CARBOHYDRATE, METAL ION AND LIPID CONTENT IN THE C R O T A L U S A D A M A N T E U S VENOM EXONUCLEASE

L U B E N B. D O L A P C H I E V , R O S I T S A A. V A S S I L E V A a n d K A M E N S. K O U M A N O V *

Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgaria) ( R e c e i v e d M a r c h 3rd, 1 9 7 9 ) (Revised m a n u s c r i p t received J u n e 1 2 t h , 1 9 7 9 )

Key words: Exonuclease; Amino acid composition; Snake venom; Carbohydrate content; Metalloenzyme ; Lipid content; (Crotalus adamanteus)

Summary Exonuclease from Crotalus adamanteus venom has only threonine as N-terminimal amino acid residue. It was examined for its amino acid composition, -SH and S-S groups. It has no free -SH groups and seven S-S bonds. The analysis of the carbohydrate residues in the enzyme proves that it is a glycoprotein. It contains neutral sugars (9.2%), amino sugars (1.9%) and ten sialic acid residues per molecule. The venom exonuclease is a metalloenzyme. This is proven by the existence of Mg 2÷, Zn 2÷ and Ca 2÷ and their specific role in the catalytic reactions. The enzyme contains also triacylglycerols (1.54%) and cholesterol esters (1.43%). The influence of the non-protein moieties of the exonuclease on its catalytic ability has been discussed. Introduction Venom exonuclease (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) is an inevitable tool for nucleotide sequence studies. But it is also a convenient model for kinetic investigations on the interaction of an enzyme with a macromolecular substrate. * Permanent address: Central Biophysical Laboratory, Bulgarian Academy of Sciences, Sofia 1113, Bulgaxia. Abbreviation: SDS, sodium dodecyl sulfate.

332 The recent development of an efficient m e t h o d for purification of the exonuclease from CrotaIus adamanteus venom (the first paper of the series [1] ) as well as the determination of some physicochemical parameters of its molecule [2] permit the chemical characterization of this enzyme, as well as some structural studies of its active site. The present paper reports the amino acid composition of the exonuclease isolated from C. adamanteus venom, the non-protein components and its N-terminal amino acid residue. Materials and Methods

Enzyme preparation. The exonuclease was isolated from C. adamanteus venom (Sigma} according to the previously described m e t h o d [1] with the following modifications: the concentration-dialysis against Ficoll 400 after the affinity chromatography and gel filtration steps was replaced b y extensive dialysis at 4°C against 0.01 M ammonium acetate buffer pH 6 and lyophilization. The homogeneity of the enzyme thus obtained was checked by SDS disc electrophoresis [1], immunoelectrophoretically (see the next paper of this series) and by determining the N-terminal amino acid residue. The extinction coefficient of the venom exonuclease was defined by the absorbance of 1 mg/ml solution (0.1, w/v) in H20 at 280 nm using Carl Zeiss (Iena) spectrophotometer. The activity of native or modified by different reagents enzyme was carried out with bis-p-nitrophenyl phosphate [1]. The kinetic investigations of the EDTA-treated enzyme and the influence of the different metal ions were performed with ATP as substrate. The course of the reactions was recorded titrigraphically as described earlier [3]. The Michaelis parameters were computed from more than six substrate concentrations by linear regression analysis. Amino acid composition. The enzyme samples were dissolved in 5.7 M HC1 and hydrolysed at l l 0 ° C under vacuum for 24, 48 and 72 h. The analysis was carried out by Bio Cal BC-200 amino acid analyzer (LKB) using two standard columns program, according to Stein and Moore [4]. The results were extrapolated for 0 h. The N-terminal amino acid was determined according to Ref. [5]. 5.1 mg enzyme were treated with diphenylindenonylisothiocyanate and the TLC of the amino acid derivatives were spotted under ultraviolet where the sensitivity of the m e t h o d is 10 -11 mol. The free -SH groups of the venom exonuclease were determined by Elman's m e t h o d [6], using dithio-bis(2-nitrobenzoic acid} (Sigma) in 8 M urea, and the course of the reaction was recorded by double-beam spectrophotometer UV-VIS Specord (Carl Zeiss, Iena) at 412 nm. The a m o u n t of the liberated thionitrobenzoate ions was calculated using 13 600 as molar extinction coefficient. The S-S bonds to be determined were first reduced by 2-mercaptoethanol. 1.1 mg lyophilized enzyme was dissolved in 1 ml 8 M urea and adjusted to pH 8.5 by Tris. After adding 2 pl 2-mercaptoethanol the sample was incubated under nitrogen for 4.5 h at room temperature. The mixture was acidified with

333 acetic acid to pH 3.7 and the reducing agent was eliminated by Sephadex G-25 column chromatography. The protein-containing fractions were brought up to pH 8 by 0.1 M Tris and to 8 M by adding solid urea. The mixture was treated with dithio-bis(2-nitrobenzoic acid) as described above. Content of metal ions. The preparation of the venom exonuclease for metal ions investigation was carried out in deionized water and all necessary precautions were taken to eliminate artificial contamination. The analysis was accomplished by atomic absorption using Pay Unicam AR-25 equipment. Determination of carbohydrate residues. The content of neutral sugar residues was determined by the phenol m e t h o d of Dubois et al. [7], using galactose (Merck) as standard. The hexoseamine content was measured by Ehrlich's reagent according to Elson and Morgan [8]. Glucosamine hydrochloride (Fluka) was used for standardization of the method. The sialic acid was determined in a sample of 0.5 ml containing 1.23 mg enzyme. The solution was mixed with an equal volume of 0.1 mg/ml 1,10phenanthroline and the a m o u n t of sialic acid estimated spectrophotometrically [9] from the absorbance at 306 nm and 3070 as molar extinction coefficient. Analysis for lipids. Using Oil Red for staining polyacrylamide gels after an electrophoretic run of venom exonuclease, it was observed that the disc, corresponding to the enzyme was stained according to Utermann [10]. The lipids in the enzyme preparation were extracted according to Folch et al. [11] and fractionated by TLC en Silica gel H plates (Merck) in the following solvent systems: CHCla/C2HsOH/acetone/H20 (70 : 35 : 8 : 4) or hexane/ethyl ether/acetic acid (90 : 30 : 1). 2',7'-Dichlorofluorescein and iodine vapors or 50% sulfuric acid were used as locating agents. The a m o u n t of the cholesterol esters was determined colorimetrically [12]. Results and Discussion The initial investigation of the carbohydrate c o n t e n t in an exonuclease isolated from C. adamanteus venom according to the previously published m e t h o d [ 1 ] showed an unacceptable a m o u n t of neutral sugars, which slightly decreased after dialysis. It was found that the concentration-dialysis against Ficoll, as proposed in the purification procedure led to contamination of the exonuclease with some low molecular weight substances (mainly carbohydrates, Ca 2÷) present in the Ficoll probably as a by product of its preparation. This necessitated the substitution of Ficoll concentration with lyophilization from 0.01 M amm o n i u m acetate during the purification and from H20, of the last product. The exonuclease thus obtained is completely free of any contaminating proteins and only threonine was obtained as an N-terminal amino acid. The extinction coefficient of the enzyme under study as defined at 280 nm was 1.04. The amino acid composition of the exonuclease is shown in Table I. The calculation of the number of the amino acid residues was accomplished on the basis of both the number of half-cystine from the amino acid analysis and from the determined S-S bonds. The venom exonuclease contains all the natural amino acids. It is poor in sul-

334 TABLE I A M I N O A C I D C O M P O S I T I O N O F T H E E X O N U C L E A S E I S O L A T E D F R O M T H E V E N O M O F C. A D A MANTEUS

A m i n o acid residue

pg a m i n o a c i d / mg protein

Residues/mol of protein *

Asx ** Thr Set Glx ** Pro Gly Ala

105.70 4.71 46.00 87.60 43.91 25.79 26.62

130 62 76 96 64 64 53

Cys Val Met ne Leu Tyr Phe Lys His Arg

9.70 31.90 13.82 37.79 135.03 38.61 48.33 63.78 24.23 51.70

14 45 15 47 169 34 47 71 25 47

Total

835.22

1059

* See in the t e x t . ** The o b s e r v e d significant a m o u n t of a m m o n i a dttring the a m i n o acid analysis and the high i s o e l e c t r i c p o i n t ( p l = 9 . 0 ) [ 1 3 ] give us g r o u n d to a s s u m e the bigger a m o u n t o f A s x and Glx t o be Asn and Gln.

fur-containing amino acids and rich in leucine, asparagine and glutamine. The ratio Lys + His + Arg/Asp + Glu is 0.63. It was observed that the exonuclease from C. adamanteus venom does not have free sulfhydryl groups (Table II). This is not in accordance with the observation of Brown and Bowless [14]. This may be caused by the enzymes being isolated from the venoms of different species of snake. But a more likely explanation is that in their case the observed decrease in the apparent specific activity of the phosphodiesterase is due to a modification of the contaminating phosphomonoesterase, bis-p-nitrophenyl phosphate having been used as substrate for the phosphodiesterase activity of the exonuclease. The estimation of the S-S bonds in the exonuclease showed that there are seven disulfide linkages (Table II) and their reduction completely inhibits the enzyme activity. The study of the carbohydrate content in the venom exonuclease showed that the pure preparation contains 9.2% neutral sugars, 1.9% aminosugars, and 2.28% (w/w) sialic acid (Table II). The strong binding of the enzyme to immobilized concanavalin A suggests that the neutral sugar residues are mainly mannosides and/or glucosides [ 15]. From the amount of the sialic acid it was found that ten residues correspond to one molecule of exonuclease. Neither the immobilization of the exonuclease on concanavalin A-Sepharose [16], nor the treatment with neuraminidase [13] change the activity of the enzyme. We consider that the carbohydrate part has no direct connection with the catalytic part of the venom exonuclease. On the other hand the determined

335 T A B L E II C H E M I C A L C H A R A C T E R I S T I C S O F T H E C. A D A M A N T E U S Molecular

weight was determined

Molecular weight • . . J Extraction coefficient Protein -SH groups S-S bonds

as b e f o r e

at 2 8 0 n m

VENOM EXONUCLEASE

[ 2]. 142 000 + 2000 1.04 (1 m g / m l ) 83.52% (w/w) 0 7

Carbohydrates Neutral sugars Aminosugars Sialic acid Metal ions Zn 2+ Mg 2+ Ca 2+ * Lipids Triacylglycerols Cholesterol esters

9.2% 1.9% 2.26% (ten residues per enzyme

molecule)

1 (atom]tool) 6 (atoms/tool) 35 (atoms/mol) 1.45% (w/w) 1.43

* See in the text.

aminosugars, the strong binding to concanavalin A-Sepharose and the change of the pI after treatment with neuraminidase proves the covalent binding o f these carbohydrates to the polypeptide chain. The number of the metal ions obtained in the venom exonuclease are shown in Table II. Notwithstanding the significant number of Ca 2÷, which could be exchanged during the elution from concanavalin A-Sepharose, one cannot exclude its participation in the active form of the enzyme. To elucidate which particular metal is native to venom exonuclease, a kinetic analysis of the influence of Mg 2÷, Zn 2÷ and Ca 2÷ on the catalytic hydrolysis of ATP was done. The determined kinetic parameters for a steady-state reaction are summarized in Table III. It is evident that after 20 h incubation in 0.014 M EDTA and 2 h treatment with excess of these metal ions, two different types of reactivation occurred. While Mg 2÷ and Ca ~÷ restored the enzyme activity mainly by changing the affinity towards the substrate (1/Kin is significantly higher), Zn 2÷ specifically changed the catalytic step of the hydrolytic reaction (k+2 is higher). As the

TABLE III K I N E T I C P A R A M E T E R S O F H Y D R O L Y T I C R E A C T I O N S , C A T A L Y Z E D BY M O D I F I E D BY E D T A E X O N U C L E A S E , A F T E R R E A C T I V A T I N G BY D I F F E R E N T M E T A L I O N S Reactivating agent

Km (× 10 -3 M)

V ( ~ m o l • m i n -1 )

1/K m (M -1 )

k+2 (s - I )

H20 * Mg 2+ Zn 2+ Ca 2+

1.60 0.55 19.6 0.73

1.76 0.80 16.70 1.4

600 1800 50 1400

4.2 1.9 40.0 6.7

F o r d e t a i l s s e e i n t h e t e x t . tz+2 ( o r k c a t ) = V / [ E ] . * Untreated enzyme with EDTA.

336

Zn 2÷ in this case does not alter the affinity of the enzyme towards the substrate and Ca 2÷ and Mg ~÷ have a weak effect on k÷2, it can be supposed that these metals have a different effect, Zn 2÷ for the catalysis, and Mg2÷ and Ca 2+ for the binding of the substrate. This observation not only proves the exonuclease to be a metaUoenzyme but conclusively distinguishes between the two different effects of the metals. In the preparation of the venom exonuclease we measured 1.54% content of triacylglycerols and 1.13% of cholesterol esters (Table II). During the first step of the purification procedure the enzyme is precipitated by 45% acetone saturation. Then it is quite unlikely that the lipids obtained are superficial contaminations. We can suppose that these lipids are bound at some hydrophobic regions of the polypeptide chain. The observed non-inhibitory effect of digitonin shows t h a t either they have no direct influence on the activity or t h e y are beyond the reach of this neutral detergent. In conclusion we can state that the exonuclease, isolated from C. adam a n t e u s venom by the previously published m e t h o d [1] has one N-terminal amino acid residue. The enzyme has quite a complex structure, it is both a glycoprotein and a metalloenzyme. There is no direct proof of the character of the binding of the lipid moieties but one cannot exclude a certain polarization of the enzyme, taking into consideration the strong hydrophility of its macromolecular substrate. A detailed analysis of the metal ions in the structure of the exonuclease is in progress. References 1 Dolapchiev, L.B., Sulkowski, E. and Laskowski, M., St. (1974) Biochem. Biophys. Res. Commun. 61, 273--281 2 Dolapchiev, L.B., Vasyl, Z. and Ostrowski, W. (1970) C.R. Acad. Sci. Bulg. 23, 1433--1436 3 Dolapchiev, L.B. (1970) Biokhimi]a 35, 1073--1077 4 Stein, W.H. and Moore, S. (1949) J. Biol. Chem. 178, 79--91 5 Ivanov, Ch. and Mancheva, I. (1973) Anal. Biochem. 53, 420--430 6 Elman, G.L. (1959) Arch. Biochem. Biophys. 82, 70--77 7 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Anal. Chem. 28, 350--356 8 Elson, L.A. and Morgan, W.T.J. (1933) Biochem. J. 27, 1824--1829 9 Dimitrov, G.D. (1973) Hoppe-Seylers Z. Physiol. Chem. 354, 121--124 10 Utermann, G. (1972) Clin. Chim. Acta 36, 521--529 11 Folch, J., Lees, M. and Sloan-Stanley, G.H. (1957) J. Biol. Chem. 226, 497--503 12 Sperry, W.H. and Webb, M. (1950) J. Biol. Chem. 187, 97--101 13 Dolapchiev, L.B., Ostrowski, W., Wasylewska, E., Wasylewski, Z., and Weber, M. (1977) Bull. Acad. Pol. Sci., 25, 359--362 14 Brown, J.H. and Bowles, M.E. (1965) US Army Med. Res. Lab. Fort Knox, KY., Rep. No. 627, 1--13 15 Poretz, R.D. and Goldstein, I.J. (1970) Biochemistry, 9, 2890--2896 16 Sulkowski, E. and Laskowski, M., Sr. (1974) Biochem. Biophys. Res. Commun., 5 7 , 4 4 3 - - 4 4 7