hu. J. Biochem. Vol. 24, No. 6, pp. 967-913, 1992

0020-71IX/92 $5.00+ 0.00 Copyright 0 1992Pergamon Press plc

Printed in Great Britain. All rights reserved

PURIFICATION AND PROPERTIES OF THE L-AMINO ACID OXIDASE FROM MONOCELLATE COBRA (NAJA NAJA KAOUTHIA) VENOM NGET-HONG TAN

andSOMASUNDARAMSWAMINATHAN

Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia [Tel. 03-750-2053; Fax 03-603-757-36611 (Received 13 August 1991) Abstract-l. The L-amino acid oxidase of the monocellate cobra (Baja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.

Determinations of protein concentration and L-amino acid oxidase activity

INTRODUCTION L-Amino acid oxidase (L-amino acid : 0, oxidoreductase, EC 1.4.3.2) catalyzes the oxidative deamination of L-amino acids (for reviews, see Bright and Porter, 1975; Iwanaga and Suzuki, 1979; Meister and Wellner, 1963). The enzyme occurs widely in snake venom. Several authors have reported the isolation and characterization of L-amino acid oxidases from various snake amino venoms (Kurth and Aurich, 1973; Shaham and Bdolah, 1973; Singer and Kearney, 1950; Sugiura et al., 1975; Tan and Saifuddin, 1989; Ueda et al., 1988; Wellner and Meister, 1960). As yet, however, no L-amino acid oxidase from cobra (genus Baja) venom has been investigated in detail. We describe here the purification and some properties of the L-amino acid oxidase from monocellate cobra (Baja nuju kaouthiu) venom. The specificity constants (k,,,/K,,,) of the enzyme for various L-amino acids and derivatives were also determined and the substrate specificity of the enzyme was investigated. MATERIALS AND

Protein concentration was determined by the Lowry method (Lowry et al., 1951). L-Amino acid oxidase activity was determined spectrophotometrically with L-leucine as substrate using the method modified from Bergmeyer (1983), as described previously (Tan and Saifuddin, 1989). One unit of enzyme activity was defined as the oxidation of 1 pm01 of L-Leu per min. Purification oxidase

of monocellate

cobra

venom L-amino acid

All the following operations were carried out at 4°C. (i) Sephadex G-200 gel filtration chromatography. One gram of crude monocellate cobra venom was dissolved in 10 ml of 0.85% sodium chloride and applied to a Sephadex G-200 gel filtration column (2 x 80cm) equilibrated with 0.02M ammonium acetate buffer, pH 7.4. Elution was carried out using the same buffer at a flow rate of IO ml/hr and fractions of 5ml were collected. The effluent was analyzed for L-amino acid oxidase activity and fractions exhibiting high L-amino acid oxidase activity (tubes 2638) were pooled. (ii) First Bio-Rex 70 cation exchange chromatography. The L-amino acid oxidase fraction obtained from step (i) was fractionated by a Bio-Rex 70 ion exchange column (2 x 14 cm) equilibrated at pH 6.0, as described by Karlsson et al. (1971). The column was washed with 0.02M ammonium acetate prior to application of sample. Following sample application, the column was eluted with the same ammonium acetate for one bed volume followed by a linear, 0.14.6M ammonium acetate gradient (600 ml to 600 ml). Fractions of 5 ml were collected and the flow rate was 30ml/hr. Fractions constituting peak 4, the protein peak that exhibited strong L-amino acid oxidase activity (tubes 16&178), were pooled. (iii) Second Bio-Rex 70 cation exchange chromatography. Peak 4 obtained in step (iii) was further fractionated by a second Bio-Rex 70 ion exchange chromatography using the

METHODS

Materials

Freeze-dried monocellate cobra (Baja naja kaourhia) venom was obtained from Southeast Asia Venom Institute (Kuala Lumpur, Malaysia). Croralus adamanteus venom was from Miami Serpentarium Laboratories (Salt Lake City, U.S.A.) Sephadex G-200, Sephadex G25 and pha&nalytes were- obtained from Pharmacia Fine Chemicals. Bio-Rex 70. minus 200 mesh was from Bio-Rad Laboratories (Richmond, U.S.A.). All other reagents and substrates for kinetics investigations are of analytical grade and were purchased from Sigma Chemical Company. 967

968

NGET-HONG TAN and S~MASUNDARAM SWAMINATHAN

same column as in (ii) but eluted by a linear gradient of 0.24.5M ammonium acetate (1000 ml to 1000 ml). Fractions of 5 ml were collected. Polyacrylamide gel electrophoresis, SDS-polyacrylamide electrophoresis and isoelectric focusing

gel

Both polyacrylamide gel electrophoresis (PAGE) and SDS-polyacrvlamide gel electrouhoresis (SDS-PAGE) were carried out -at pH 8.4 with i2.5% polyacrylamide gel (Laemmli, 1970). The isoelectric point of the L-amino acid oxidase was determined using an electrofocusing column of 110 ml capacity (Vesterberg and Svensson, 1966). Determinaiions of molecular weight and amino acid analysis The molecular weight of the purified enzyme was determined by: (i) Sephadex G-200 gel filtration chromatography using catalase (mol. wt 232,000), alcohol dehydrogenase (mol. wt ISO,OOO), bovine serum albumin (mol. wt 66,000) pepsin (mol. wt 34,000) and myoglobin (mol. wt 17,200) as standard proteins; and (iii) SDS-PAGE (see above) using bovine serum albumin (mol. wt 66,000), ovalbumin (mol. wt 45,000), glyceraldehyde-3-phosphate dehydrogenase (mol. wt 36,000), trypsinogen (mol. wt 24,000) soybean trypsin inhibitor (mol. wt 20,100) and a-lactalbumin (mol. wt 14,200) as marker proteins. The purified enzyme was hydrolyzed in 6M boiling hydrochloric acid in sealed tube at 1IO”C for 24 hr and analyzed by a Beckman-Spinco amino acid analyzer. Half-cystine was determined according to Hirs (1967). Tryptophan was not determined. Stability studies The purified enzyme (O.O5mg/ml) in appropriate buffer was filtered through a milhpore filter (0.22 pm) and stored in a sterilized vial and incubated at various temperatures. Aliquots (50~1) were removed at various time intervals for assay. To investigate the effect of freezing, the purified enzyme (0.05 mg/ml, in 0.85% saline) was quick frozen with dry ice/acetone mixture and then stored at -20°C for 60 hr. For comparison, a Crofalus adamanteus venom sample (1 mg/ml) was treated similarly. Kinetics The kinetic parameters K, and k,,, were determined at 25°C using Lineweaver-Burk reciprocal plots. All the kinetic parameters determined herewith are a function of oxygen concentration in the reaction mixtures. Under the experimental conditions (uide infia), oxygen concentration was approximately constant as it was estimated that throughout the initial rate measurement period, less than 5% of dissolved oxygen (approx 0.24mM, see Bright and Porter, 1975) was consumed. Rate measurements were carried out with a Hiatchi 150 UVVIS recording spectrophotometer. The reaction mixture consisted of 0.05 ml purified enzyme (2-7 pg), 0.05 ml of 0.0075% horseradish peroxidase (100 purpurogalin units/mg), 67.5 pg 0-dianisidine and appropriate amount of substrate in 0.9 ml of O.lM Tris-HCl, pH 8.5 or other appropriate buffers when specified, and the initial rate was measured as the increase in absorbance at 436nm. The difference in molar absorption coefficient is 8.31 x lO’M_’ cm-’ (Bergmeyer, 1983). Estimation of incremental binding energies of the side chains of L-amino acid substrates lo the en:yme The incremental binding energy of a particular group on a substrate molecule is defined as the contribution that group makes to the Gibbs free energy of transfer of the

molecule from aqueous solution to the binding pocket of the protein (Fersht, 1985; Fersht et al., 1980). The incremental binding energy of a group R on a substrate R-S, relative to the hydrogen on the smaller substrate H-S may be estimated

by the following

equation

(Fersht

et al., 1980):

A& = - RT ln (k,,lKJ,-J(k,,, IKm)H-s where R is the universal gas constant and T the absolute temperature. In this estimation, the electronic inductive effect of R on the reaction is ignored. Temperature was 25°C. In the present study, the incremental binding energy of the side chain of L-amino acid is defined as relative to the methyl group of L-alanine. Estimation of binding energy between certain part of the side chain of ~-amino acid and the appropriate binding subsite qf lhe enzyme The binding energy between certain parts of the side chain of L-amino acid substrate and the appropriate binding subsite of the enzyme was estimated by the difference in incremental binding energies of the side chains of appropriate L-amino acids, as described previously (Tan and Saifuddin, 1991). RESULTS

PuriJication of the L-amino cellate cobra venom

acid oxidase from

mono-

Sephadex G-200 gel filtration chromatography of the crude monocellate cobra venom (1 g, LAAO activity 0.0385 unit/mg) yielded two major protein peaks, peak 1 and 2, and the L-amino acid oxidase activity occurred between the two peaks (Fig. 1). Fractions exhibiting L-amino acid oxidase activity were pooled and fractionated by a Bio-Rex 70 cation exchange chromatography. Six protein peaks were obtained with L-amino acid oxidase activity occurring mainly in peak 4 (Fig. 2). Peak 4 was then further fractionated by a second Bio-Rex 70 chromatography. Only one protein peak which exhibited L-amino acid oxidase activity was obtained (Fig. 3). This preparation was designated as the purified monocellate cobra venom L-amino acid oxidase. It had a specific activity of 4.59 unit/mg, representing a 119-fold of purification. Characterization

of the purified

enzyme

The purified monocellate cobra venom L-amino acid oxidase migrated as a single band in both the

polyacrylamide and SDS-polyacrylamide gel electrophoresis, indicating that it is homogeneous electrophoretically. The enzyme had an isoelectric point of 8.12 and molecular weight of 112,200 as determined by Sephadex G-200 gel filtration chromatography and 57,400 as determined by SDS-polyacrylamide gel electrophoresis in the presence of /I-mercaptoethanol. Amino acid analysis showed that the purified enzyme contained (mol %): Ala (6.76%), Arg (5.91%) Asx (10.62%) Glx (10.45%) Cys/2 (2.98%) Gly (8.97%) His (2.24%) Ileu (5.26%) Leu (7.38%) Lys (6.38%) Met (1.30%) Phe (3.26%) Pro (6.12%) Ser (7.64%) Thr (5.64%) Tyr (4.19%) and Val (4.89%). Tryptophan was not determined. The purified monocellate cobra venom L-amino acid oxidase exhibited remarkable thermal stability. The enzyme retained 100% activity after incubation at 25 and 4°C for 2 weeks. The enzyme was also rather stable in alkaline medium, retaining full activity after incubation for 1 hr at pH 9.0 (O.lM Tris-HCI), at 25°C. Under the same experimental conditions, the L-amino acid oxidase activity of Crotalus adamanteus venom lost 60 and 85% of

Snake venom L-amino acid oxidase

-

969

2.5-

z 3 2.0z w 1.5Y 2 a 1.0. 2 a

0.5 -

,b

20

30

40

TUBE

50

60

70

8

NUMBER.

Fig. I. Fractionation of monocellate cobra venom by Sephadex G-200 gel filtration chromatography. One gram of the venom was applied to the column (2 x 80 cm) equilibrated with 0.02M ammonium acetate buffer, pH 7.4. Tube volume = 5 ml.

activity, respectively, after 10min and 1 hr incubation. The purified monocellate cobra venom enzyme, however, lost 21% of activity after storage at -21 “C for 60 hr. Under the same experimental conditions, L-amino acid oxidase from Crotalus adamanteus venom lost all enzymatic activity. Kinetic studies The catalytic oxidation of all reactive substrates by monocellate cobra venom L-amino acid oxidase followed Michaelis-Menten kinetics at 25”C, with substrate inhibition evident at high concentration. Figure 4 shows the Lineweaver-Burk plot of the oxidation of L-Leu by the purified enzyme at pH 8.5 (O.lM Tri-HCI buffer). Substrate inhibition was

20

40

60

130 140 TUBE

160

evident when the concentration of L-leucine exceeded 3mM. The pH profile of the purified monocellate cobra venom L-amino acid oxidase with L-Leu as substrate is shown in Fig. 5. The purified enzyme exhibited optimum V,.,/K,,, value at pH of 8.5 and it is noted that the enzyme was still very active even at the very alkaline pH of 10. There was a slight buffer effect: the V,,,/K,,, values in 50 mM sodium phosphate buffer were slightly higher than those in 50mM Tris-HCl buffer (Fig. 5). Table 1 shows the values of k,,,, K,,, and k,,,/K, of the oxidation of the various L-amino acids and derivatives by the purified monocellate cobra venom L-amino acid oxidase at 25”C, in O.lM Tri-HCl

180

200

220

240

NUMBER

Fig. 2. Bio-Rex 70 cation exchange chromatography of L-amino acid oxidase fraction obtained from Sephadex G-200 gel filtration. The Bio-Rex 70 column (2 x 14cm) was equilibrated with 0.02M ammonium acetate buffer, pH 6.0. A 0.1-0.6M ammonium acetate gradient (600 ml to 600 ml) was used. Tube volume = 5 ml.

970

NGET-HONG TAN and SOMASUNDARAM SWAMINATHAN

8

$ g

x

E

4

E

I I I I I

.E E

0.04-

0

-0.6

?

.0.5

-0.08

z5

-0.4

;5 dE

-0.06

5

a.3

a

-0.04

0

.0.2

-0.02

3

.o .l

.3

ti f$o.o2P O.Ola

20

40

80

60

100 TUBE

200 NUMBER

220

240

260

I

E

G 0.03-

260

Fig. 3. Second Bio-Rex 70 chromatography. Pooled fractions with L-amino acid oxidase activity obtained from the first Bio-Rex 70 chromatography were loaded onto the column (2 x 12 cm) equilibrated with 0.02M ammonium acetate buffer, pH 6.0. The gradient used was 0.2&5M ammonium acetate (1000 ml to 1000 ml). Tube volume = 5 ml. Insert: (A) PAGE of the purified enzyme; (B) SDS-PAGE of the purified enzyme; (C) SDS-PAGE of the molecular weight markers. The seven bands correspond to: (1) bovine serum albumin; (2) ovalbumin; (3) glyceraldehyde-3-phosphate dehydrogenase; (4) carbonic anhydrase; (5) trypsinogen; (6) soybean trypsin inhibitor; (7) a-lactalbumin. The proteins migrated from cathode (top) to anode (bottom).

buffer, pH 8.5. o-Leucine, o-methionine, L-cysteine, glycine, L-serine, L-threonine, L-glutamic acid, Laspartic acid, L-prohne, L-2,4-diamino butyric acid and L-2,3-diamino propionic acid were not oxidized. For the other L-amino acids tested, the K,,, values ranged from 0.023 mM (L-tyrosine) to 61.35 mM (L-alanine), k,,, values ranged from 1.25 set-’ for L-valine to 25.71 set-’ for L-arginine. The specificity constants k,,,/K, for the substrates examined varied about 2400-fold, with L-phenylalanine being most effectively oxidized while L-valine was least effectively oxidized.

Using the specificity constant as a criterion, the enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline; slightly active against L-histidine, L-cystine, L-amino butyric acid and L-isoleucine; L-glutamine, L-lysine, L-asparagine, L-ornithine, L-alanine and L-valine were oxidized very slowly. Table 2 shows the estimated incremental binding energies of the various side chains (R) of L-amino acids to the enzyme relative to R = CH3 (Ala).

Vmax

/Km

(min)

-1

18 16 14 12 10 8 6

-l I

0.4

I

I 0.8

+

1.2 (mM

I 1.6

I

I

2.0

1-l

Fig. 4. Lineweaver-Burk plot for the oxidation of L-leucine by the purified monocellate cobra venom L-amino acid oxidase. The reactions were carried out in O.lM Tris-HCl buffer, pH 8.5 and at 25°C and the reaction mixture contained 2.4 pg enzyme.

Fig. 5. The effect of pH on the specificity constant of purified monocellate cobra venom L-amino acid oxidase using L-leucine as substrate. The reactions were carried out in either 0.05M sodium phosphate buffer (O-0, pH 5-7) or 0.05M Tris-HCl buffer (a---0, pH6.5-10) and at 25°C. The reaction mixture contained 0.8 pg enzyme.

Snake venom L-amino acid oxidase Table

I.

Kinetic

parameters

of monocellate acid oxidase”

cobra venom

t_-amino

acid

16.67 61.35 43.50 11.59 26.67 I8.60 5.00 12.90 2.86 5.49 2.27 I .69 0.82 0.66 0.63 0.29 0.023 0.06

1.25 6.23 4.91 1.79 6.39 4.91 3.58 9.35 3.04 9.74 14.10 25.71 21.04 23.38 24.39 12.41 3.21 IO.75

75.0 101 .s 112.9 154.4 239.6 264.0 716.0 724.8 1062.9 1774. I 6211.5 15,213.O 25,658.S 35.424.2 3g714.3 43,000.0 142.173.9 179.166.7

‘The

reaction mixture consisted of 0.05ml (2.4gg) purified enzyme, 0.05 ml of peroxidase (0.0075%). 67.5 pg O-dianisidine and appropriate amount of the substrate in 0.9ml of 0.1 M Tris-HCI buffer, pH 8.5 and the kinetics experiments were conducted at 25’C. bThe following amino acids and derivatives were not oxidized by the purified t-amino acid oxidase: D-leucine, o-met&urine, t.-cysteine, glycine, r-swine, L-threonine, tglutamic acid, Laspartic acid, r-proline, t-2,4-diamino n-butyric acid and L-2,3diamino propionic acid.

DISCUSSION

Pur$cation and characterization venom ~-amino acid oxidase

of monocellate cobra

Monocellate cobra venom t-amino acid oxidase was purified to electrophoretic homogeneity with a purification fold of 119. The chromatographic profiles indicate that there is only one form of L-amino acid oxidase in the venom. Many authors have reported the presence of multiple forms of L-amino acid oxidase in some snake venoms (Hayes and Wellner, 1969; Nakano et al., 1972; Shaham and Bdolah, 1973). On the other hand, venoms of TrimeresurusjZavoviridis and T. mucrosquumatus have been demonstrated to exhibit only a single form Table 2. Incremental

of L-amino acid oxidase (Nakano et al., 1972; Ueda et al., 1988).

Substrateb t.-Valine L-Alanine t-Omithine t.-Asparagine t-Lysine L-Glutamine L-Isoleucine r.-Amino-butyric t_-Cysteine t,-Histidine t.-Norvaline t.-Arginine t-Norleucine t.-Leucine t.-Methionine t-Tryptophan t.-Tyrosine t_-Phenylalanine

971

The purified L-amino acid oxidase had a molecular weight of 112,200 as determined by Sephadex G-200 gel filtration and 57,400 as determined by SDS-polyacrylamide gel electrophoresis, suggesting that this enzyme, like many other venom L-amino acid oxidase, is also composed of two subunits with comparable molecular weight. The moiecular weights of other snake venom L-amino acid oxidases range from 60,000 to 140,000 (see Iwanaga and Suzuki, 1979). Like most other venom L-amino acid oxidase, the purified monocellate cobra venom L-amino acid oxidase was stable at room temperature and at 4°C but was partially inactivated when stored in frozen form. The enzyme, however, exhibited remarkable stability at alkaline pH medium. Many venom L-amino acid oxidase was very unstable at alkaline pH (Paik and Kim, 1966; Ueda et al., 1988). The purified enzyme exhibited a pH optimum of pH 8.5 with t.-leucine as substrate. L-amino acid oxidases from Ophiophag~s h~nah, ~gk~trodon caiiginosus and Vipera palaestinae venoms also exhibited similar range of pH optimum (Shaham and Bdolah, 1973; Sugiura et al., 1975; Tan and Saifuddin, 1989). On the other hand, L-amino acid oxidases from Crotalus adamanteus, Vipera ammodytes and Trimeresurus mucrosquamatus venoms had a pH optimum of 7 to 7.6 (Wellner and Meister, 1960; Ueda et al., 1988). It should be noted, however, that the shape of the pH profile and hence the pH optimum of L-amino acid oxidase also depends on the type of amino acid substrate used. For example, it has been reported that for Crotalus adamante~s venom L-amino acid oxidase, six different pH-profiles were obtained when different substrates were used (Paik and Kim, 1965). Substrate

speciJicity

Monocellate cobra venom L-amino acid oxidase did not oxidize D-leucine and D-methionine, while the corresponding t-enantiomers were readily oxidized, confirming that the enzyme is specific for the

bmdmg energies of side chains of some t-amino acids to monocellate acid oxidase (relative to t_-alanioe)

k,,,ltu, (sec.‘M

Substrate t-Amino

acids and derivatives with general structure t_-Atanine R=CH, t.-a-Amino-n-butyrtc acid R = CH,CH, t_-Norvaline R = CH:CH,CH, t_-Norleucine R = CH,CH,CH,CH, I.-Valine R = CH(CH,), L-lsoleucine R = CH(CH,)CH,CH, t.-Leucine R = CH,CH,CH(CH,), t-2,3 Diamino-propionic acid R = CH*NH, L-2,4 Diamino-n-butyric acid R = CHrCH,NH, L-Ornithine R = CH,CH,CH,NH, I>-Lysine R = CH,CH,CH,CH,NH, L-Arginine R = CH,CH,CH,NHC(NH)NH, L-Phenylalanine R = CH,C,H, t,-Tyrosine R = CH,C,H,OH L-Tryptophan R = CH,-indole L-Methionine R = CH,CH,SCH, L-Histidine R = CH&nidazole ‘This refers to the incremental

binding

cobra venom t-amino

‘f

NH,CHRGCO,H 101.5 724.8 6211.5 25658.5 75.0 716.0 353424.2 0 0 112.9 239.6 15,213.O 179.166.7 142.173.9 43,000.0 38,714.3 1774. I

free energy of side chain of the amino acid relative

incremental binding energy” (keal/~ol) 0 - I.16 - 2.44 - 3.28 +0.1X -1.16 -3.47

-0.06 -0.51 -2.91 -4.43 -4.29 -3.58 - 3.52 -1.69 to r-alanine

NGET-HONGTAN and SOMASUNDARAMSWAMNATHAN

972

L-enarbmer of amino acid. L-Proiine was not oxidized, this is in agreement with previous observations that effective oxidation of L-amino acids by L-amino acid oxidase requires the presence of a primary a-amino group (Iwanaga and Suzuki, 1979; Meister and Weliner, 1963; Tan and Saifuddin, 1991). The side chain specificity of many snake venom L-amino acid oxidases have been investigated (see Iwanaga and Suzuki, 1979; Tan and Saifuddin, 1991) and the results seem to indicate some species difference. Unfortunately, most side chain specificity studies were conducted under rather arbitrary substrate concentrations and the results are therefore of limited significance. For king cobra venom t-amino acid oxidase, however, comparison of the specificity constants of the enzyme for various substrates suggested the presence of an amino acid “side chain binding site” in the enzyme, and that the binding site comprises at least five subsites, including three hydrophobic subsites and two “amino” binding subsites. In the present study, Tables I and 2 provide quantitative information concerning the amino acid side chain specificity of monocellate cobra venom t-amino acid oxidase. Our data clearly show an improvement in the specificity constant which results from additional hydrophobic interaction between the hydrocarbon of the side chain R and the binding site. as the size of the side chain increases from R = CH, to R = n - C, H,. The data also suggest that branching at the a-carbon is undesirable but branching at the y-carbon can be accommodated. Thus, L-valine was a very poor substrate, and L-norvaiine was a much better substrate than L-isoieucine, while Lthreonine was not oxidized. On the other hand, L-leucine and L-norieucine were both good substrates with comparable specificity constant. These data suggest that the monoceilate cobra venom L-amino acid oxidase also possesses an aikyl binding site for the binding of the side chain of the amino acid substrate, and that the binding site comprises at least four subsites, each accommodating a methylene~methyl carbon. The four subs&es are henceforth termed subsites a, b, c and d, respectively, which binds the beta, gamma, delta and epsiion carbons of the side chain of r_.-norleucine (Fig. 6. bottom). Subsite a apparently can only accommodate one methyiene/methyt carbon, as branching at the

b

auc

d b

Fig. 6. A tentative model for the side chain binding site of monocellate cobra venom L-amino acid oxidase; showing (top) the binding of the side chain of r;-leucine to the binding site; and (bottom) the binding of the side chain of L-norleucine to the binding site,

P-carbon is undesirable; while subsite b can accommodate two methylene/methyI carbons (Fig. 6, top, shows the binding of the side chain of r,-leucine to the proposed binding site). Incremental energy calculations indicated that the binding energies between subsite c and d and the methylene/methyl carbons were - I .28 and -0.84 kcai/mol, respectively, and between subsite b and the two methylene/methyl carbons were - 1t 16 and - 1.03kcai/mol, respectively. On the other hand, binding of subsite b, c or d by a polar group such as a primary amino group rest&s in destabilization, as shown by the fact that L-2,3-diamino propionic acid, L-2,4-diamino n -butyric acid and r,-ornithine were all very poor substrates (see also Table 2). These observations suggest that the three subsites b, c and d are all hydrophobic in character. It is, however, not possible to estimate the binding energy between subsite a and methylene/methyl carbon as the enzyme did not oxidize glycine at all. It is interesting to note that L-lysine was poorly oxidized by the enzyme, suggesting the possible existence of an additional subsite that is also probably of hydrophobic character. It is also noted the binding site can bind the side chain of L-arginine well (Table 2). It is not known whether the guanidino group binds to subsite c only or also to subsite d or some other unidentified subsites. The data also show that cyclic structures such as phenyl, phenol or indole group can be accammodated in the side chain binding site of the enzyme presumably in a mode similar to the binding between the side chain of L-leucine and the enzyme, in the manner described earlier for king cobra venom Lamino acid nxidase (Tan and Saifuddin, 1991). It is noted that aromaticity in the side chain may have some advantage for binding to the binding site of monoceliatc cobra venom L-amino acid oxidase, as indicated by the fact that L-phenylalanine and t-tyrosine were both much more effectively oxidized by L-ieucine (Table 2). The proposed side chain binding site model is consistent with the observed specificity constants for most substrates investigated in this work. Thus, L-methionine was a good substrate as it has a hydrophobic sulfur atom binding the hydrophobic subsite c. On the other hand, L-histidine was a rather poor substrate as the binding of the amino acid to the enzyme probably will place a secondary amino group on the hydrophobic subsite b. L-Amino acids or derivatives which have polar or charged group in their side chains (including L-serine, L-cysteine, L-aspartic acid, r_-glutamic acid, t_-asparagine and t,-glutamine) were poorly oxidized or not oxidized. Thus, while both monoceilate cobra venom and king cobra venom L-amino acid oxidases possess an afkyl side chain binding site there is some differences in the properties of the binding site: the alkyl side chain binding site of king cobra venom L-amino acid oxidase comprises at least five subsites (Tan and Saifuddin, 199 I ): including three hydrophobic subsites and two “amino” binding subsites, while the binding site of monoceiiate cobra venom ~-amino acid oxidase comprises at least four hydrophobic binding subsites but definitely no amino binding site is present. These differences account for the

Snake venom

L-amino

in reactivity of the two enzymes towards L-lysine and L-ornithine: these two basic L-amino acids were both very good substrates for king cobra venom L-amino acid oxidase but were poor substrates for the monocellate cobra venom enzyme. The present work therefore demonstrates unequivocably that there is indeed species difference in the substrate specificity of snake venom L-amino acid oxidase. differences

Acknowledgements-This grant PJP 87/89 from Lumpur, Malaysia.

work was supported by a research the University of Malaya, Kuala

REFERENCES

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Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom.

1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of t...
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