BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL. Qing-Xi Chen ~, and Hai-Meng Zhou*

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1 Vol. 46, No. 2, October 1998 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL Pages An Essential Lysine Residue of Green Crab (Scylla Serrata) Alkaline Phosphatase Qing-Xi Chen ~, and Hai-Meng Zhou* ( Department of Biological Science and Biotechnology, Tsinghua University, Beijing , People's Republic of China) Rcccivcd May 10, 1998 SUMMARY The values of pk,, (10.38) and Hion (10.92 Kcal/mol) have been determined for the ionizing groups controlling activity of green crab alkaline phosphatase. The results suggest that -N-2 of lysine residue responsible for the ionzation with pk~ = and AH~ = Kcal/mol is in the active site of the enzyme. Modification of lysine residues of the enzyme by an excess of 2,4,6- trinitrobenzenesulfonic acid leads to complete inactivation. The two results coincide with each other. Quantitative assessment of the data indicates that among the reactive -NH2 groups modified only one is essential for the activity of the enzyme. Key words: Alkaline phosphatase; Lysine residue; Chemical modification; Essential residue NTRODUCTON Alkaline phosphatase (EC ) is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters q. The X-ray crystal structure of bacterial alkaline phosphatase has been recently reported to 2.0 A resolution in the presence of inorganic phosphate 121. The active site is a pocket containing a tight cluster of two zinc ions (3.9 A separation) and one magnesium ion (5 and 7 A from the two zinc ions). Alkaline phosphatase from green crab (Scylla Serrata) is also a metalloenzyme containing zinc and magnesium ions, and the structure of its active site is probably similar to that of bacterial alkaline. t is known that Cys residues are non-essential for the activity of the enzyme, but the Trp residue is essential for activity and is situated at the active site of the enzyme 131 Recently, we reported that the aginyl residue is also essential for activity and is also situated at the active site of the enzyme TM, However, the role of the lysinyl residues of the enzyme #Present address: The state and SEDC Laboratory for Tumor Cell Engineering, Xiamen University, Xiamen , People's Republic of China. Abbreviations: pnpp, p-nitrophenyl phosphate; TNBS, 2,4,6-trinitrobenzenesulfonic acid *To whom correspondence should be addressed /98/ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved

2 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL has been little explored. The result in this paper shown that the lysine residues are modified by 2,4,6- trinitrobezenesulfonic acid (TNBS) and that one of them is essential for the catalytic activity of this enzyme. MATERALS AND METHODS The alkaline phosphatase was prepared from green crab (scylla serrata) viscera according to the method of Chen et al. TM to the step of ammonium sulfate fraction. The crude preparation was further chromatographed by ion-exchange with DEAE-cellulose (DE-32), then by gel filtration through Sephadex G-150 followed by DEAE-Sephadex A-50. The final preparation was homogeneous on polyacrylamide gel isoelectric focusing electrophoresis and on HPLC chromatography. The specific activity of the purified enzyme was 3320 u/mg P- Nitrophenylphosphate (pnpp) was from E. Merck; 2,4,6-trinitrobenzenesulfonic acid (TNBS) was a Sigma product; DEAE-cellulose (DE-32) was from Whatman; Sephadex G-150 and DEAE- Sephadex A-50 were Pharmacia products. All other reagents were local products of analytical grade. Enzyme concentration was determined as described by Lowry 16j. Enzyme activity was measured as previously described by Yan and Chen [7] 10/al of the enzyme solution was added into 2.0 ml of the reaction mixture containing 2 mm p-niotrophenylphosphate, 2 mm MgC2 and 0.05 M Na2CO3 buffer, ph After reaction for 10 min at 37~ 2 ml of 0.1 M NaOH was added into the reaction mixture to stop the reaction. The enzyme activity was calculated by the increased absorption of the reaction mixture was determined at 405 nm using a molar absorption coefficient of 1.73 x 104 m-lcm -1. Absorption measurements were recorded using a Beckman DU-8B spectrophotometer. The lysine residues of the enzyme were modified in 0.1 M Tris-HCl buffer (ph 8.5) with an excess of TNBS. At different time intervals, 20 ~1 portions were taken for activity determination in 2 ml of reaction mixture. RESULTS AND DSCUSSON Determination of the pk. value of ionizing groups at the active site of the enzyme n the present study, the ph was held at different constant values, while the substrate concentrations were varied, permitting measurement of the effect of increasing substrate concentration [S] on the initial rate v. Plots of l/v versus 1/[S] for different ph conditions yield a family of straight lines with a common intercept on the ordinate and different slopes, Fig. l. The intercept on the 1/v axis is equal to l/~max while the slope is equal to Kv. The data shows that fm.~ is unchanged for ph Thus, for the ph range employed in the present study. H ~ is a competitive activator, however, increasing [H'] caused Km to decease. The apparent Km for the enzyme catalytic reaction under different ph conditions can be obtained from the data in Fig. 1. Fig2 shows the plot ofpkm versus ph. The two tangent lines (broken lines) with slopes of 0 and - 1 cross on a point which gives the value of the ionization constant, pk., as the abscissa of the point of intersection. For the data in these tests, pk,, is equal to 10.38, Table. 226

3 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL ( ~_ "i/s (lnm) "1 Fig. 1. Effect of ph on the initial value v. Final concentration of the enzyme was pm. The assay reaction was carried out in 0.05 M NaCO3/NaHCO3 buffer at different ph values at 37~ The ph values for curves 1-6 were 10.72, 10.54, 10.36, 10.20, and 10.00, respectively e~ ! t J ph Fig2. Effect of ph value on the Michaelis-Menten constant for the hydrolysis ofpnpp. taken from Fig. 1. Data 227

4 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL Table 1. Parameters of the essential lysine residue ofpenaeus penicillatus acid phosphatase. onization constant (pka) Enthalpy change(ah ~ io.) Rate constant for reaction of the enzyme with TNBS (k2) Kcal/mol 7.10 Mlmin 1 Effect of temperature on pko for the ionizing groups of the enzyme active site t is well known that the values ofpk,, for e-nh3 ~ of lysine residues in protein molecules range from and that the values ofpk~, for phenol-oh of tyrosine residues range from However, the value ofpk~, shown in Table 1 is 10.38, indicating that the ionization group may be the side chain of the lysine residue or tyrosine residue. Further measurement were then made to determine, t is necessary to ascertain properties of this ionization group at the active site of the enzyme, the effects of temperature and organic solvents on the pk, value of the ionization group at the active site. The pk,, value of the ionization groups at the active site of the enzyme were measured at different temperatures (37-50~ The Van't Hoffequation d(lnk)/d]' AH~ 2 can be used to give the the following equation pk,, - constant + (AH~ where AH is enthalpy change at standard conditions, R is the ideal gas constant (1.98 Calmol.deg- 1), and T is the absolute temperature. A plot ofpk, versus 1/]" gives a straight line with slope AH~ Fig.3. AH ~ equal to Kcal/mol, Table 1, can be obtained from the slope of the straight line. The vale of AH ~ for e-nh3 + of lysine residues ranges from Kcal/mol which the value of AH ~ for phenol-oh of tyrosine residues is 6 Kcal/mo118l. The AH ~ (10.92 Kcal/mol) obtained from the present investigation is closer to the value of the ionization constant for e-nh3 + of lysine residue of the enzyme. Therefore, this result suggests that the e-nh[ of the residue is an ionization group at the active site of green crab alkaline phosphatase, t is also known that organic solvents can inhibit ionization of the neutral acids, but do not affect the ionization of cationic acids ES. The effect of dioxan concentration on the pk, values shows that the pk, value of the ionization group of the enzyme is independent of the dioxan concentration. This result suggests that the ionization group is a cationic acid, which is consistent with it being the ~-NH3 + of the lysine residue at the active site of the enzyme. Both results from the data on the effects of temperature and organic solvent on pk,, suggest that this ionization group is ~-NH3 + of the lysine residue, and that it is situated at the active site of green crab alkaline phosphatase. Moreover, the above results also suggest that the enzyme is active 228

5 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL / 1{).2 0. /!!! FF( ()~K ~) 3.3 Fig.3. nfluence of temperature on the dissociation constant (pk,,) of the ionization group of green crab alkaline phosphatase. Assay conditions were as for Fig. 1 except for the temperature. only when the e-amino group is in cationic form (i.e. e-nhf) Therefore, a possible reaction model can be shown as E+H + EH + + S- -EH+S beh + + p where E, S and P denote enzyme, substrate and product, respectively. The enzyme is active when pn < and inactive or less active at higher ph Kinetics of inactivation of green crab alkaline phosphatase by 2,4, 6-trinitrobenzenesulfonic acid n the present investigation, TNBS specifically modified the s-nhf group of lysine residues of the enzyme molecules, and led to complete inactivation of the enzyme. Semilogarithmic plots of the remaining activity versus time at different concentrations of TNBS give a family of straight lines, Fig.4, indicating that the modification follows a pseudo-first-order reaction. The slopes of the lines are given by kl = 0.693/T1/2, where kl is the rate constant for the pseudo-first-order reaction at different concentrations of TNBS and Tv2 is the time for the half-reaction of inactivation. A plot of kl versus [TNBS] gives a straight line which passes through the origin, Fig5, showing that the inactivation rate constant kl depends on the TNBS concentration. The second-order rate constant k2 = 7.10 Mqmin - (Table 1), can be obtained from the slope of the straight line in Fig.5. According to the equation described by Hollenberg et al. 191, kl : k2[]" 229

6 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL 1 O0 80 6O > 40 < l0 ' time (min) Fig4. Semilogarithmic plots of inactivation of green crab alkaline phosphatase in the presence of different concentrations of TNBS. Experimental conditions were as for Fig. 1. Final concentration of the enzyme was 7.5 /.tm Concentrations of TNBS for curves 1-4 were 4, 6, 8 and 10 mm, respectively...,2 // i TNBS] (ram) Fig.5. Plot Ofkl versus [TNBS]. Data taken from Fig4. 230

7 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL i ( ogltnbs] Fig6 Plot of log (kl) versus log [TNBS]. Data taken from Fig.4. or log k~ - log k2 + n log [] where k2 is the second-order rate constant for the inactivation reaction of the enzyme with TNBS, [] is the concentration of the inhibitor (TNBS), and n is the molecular number of TNBS binding per enzyme molecule. A plot of log kz versus log [TNBS] gives a straight line with n equal to the slope of the straight line. The data gives n equal to 1.04 (~ 1), indicating that the modification of the lysine residue led to inactivation of the enzyme and that only one lysine residue is essential for the activity of the enzyme. REFERENCES 1. MoComb, R. B., Bower, G. N. and Posen, S., Alkaline Phosphatase, Plenum Press, New York, Kim, E. E. and Wyckoff, H. W. (1991) J. Mol. Biol Chert, Q. X. and Yah, S. X (1986) J. Xiamen Univ. 25 (5), (in Chinese). 4. Xie, W. Z, Wang, H. R., Chen, Q. X. and Zhou, H. M. (1996) Biochem Mol. Biol. Mol. nt., Chen, X. Q, Zhang, W., Zheng, W. Z., Zhao, H., Yan, S. X., Wang, H R. and Zhou, H. M (1996) J. Protein Chem., 15, Lowry, O. H. (1951) J. Biol. Chem. 193, Yan, S. X. and Chen, Q. X. (1986) J. Xiamen Univ. 24(3) (in Chinese). 8. Xu, G.J. (1983) Enzyme Action and Mechanism, Science Press n China, Beijing, p92. (in Chinese). 9. Hollenberg, PF. (1971) J. Biol. Chem. 246,