Vol. 45, No. 3, July 1998 Pages 465-473 The Effects of N-thiophosphoryl Amino Acids on the Activity of Green Crab (Scylla Serrata) Alkaline Phosphatase Qing-Xi Chen 1'3, Hai-Yan Lu 2, Chun-Ming Zhu 1, Hai-Ning Lin 3 and Hai-Meng Zhou 1'* ( Department of Biological Science and Biotechnology; Tsinghua University 2 Department of Chemistry, Tsinghua University, Beijing 100084; 3 Department of Biology, Xiamen University, Xiamen 361005, P. R. China; ) Received February 15, 1998 SUMMARY Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzed the nonspecific hydrolysis of phosphate monoesters. In the present paper, the effects of several N-thiophosphoryl amino acids on the activity of green crab alkaline phosphatase have been studied. The results show that these derivatives of amino acids can lead to reversible inactivation. The equilibrium constants for inhibitors binding with the enzyme and/or the enzyme-substrate complexes have been determined. The obtained results show that both N-thiophosphoryl-Cys and N-thiophosphoryl-Glu were non-competitive inhibitors, while other five N-thiophosphoryl amino acids were un-competitive inhibitors. For the un-competitive inhibitors, the inhibition strength follows the order N-thiophosphoryMle > -Val > -Lys > -Ala > -Tyr. Compared with respective free amino acids, it can be seen that N-thiophosphorylation of the amino acids increased their inhibition strength except the N-thiophosphoryl-Cys. Key words: Alkaline phosphatase; N-thiophosphoryl amino acids; Inhibition; Inactivation INTRODUCTION Alkaline phosphatases (EC 3.1.3.1) are widely distributed in nature and are characterized by a high ph optima and a broad substrate specifici@ '21. Alkaline phophatase is a zinc-containing metalloenzyme which catalyzes the transfer of phosphate groups to water (hydrolysis) or alcohol (transphosphorylation) using a wide variety of phosphomonoesters. The enzyme from E. call has been extensively studied 31. Recently, the X-ray crystal structure of bacterial alkaline phosphatase has been reported to 2.0 A resolution in the presence of inorganic phosphate f41. The active site is a tight cluster of two zinc ions (3.9 A separation) and one magnesium ion ( 5 and 7 _ from the two zinc ions). Alkaline phosphatase from green crab (Scylla Serrata) is also a dimeric metalloenzyme Abbreviations: ALP, Alkaline phosphatase; pnpp, p-nitrophenyl phosphate; EPS, N-thiophosphryl * To whom correspondence should be addressed. 465 1039-9712/98/09046539505.00/0 Copyright 9 1998 by Academic Press Australia. All rights of reproduction in any form reserved.
containing zinc and magnesium ions, and the structure of its active site is probably similar to that of bacterial alkaline phosphatase. It is well known that green crab alkaline phosphatase was inactivated by EDTA, and the complete kinetic course of EDTA inactivation, monitoring the hydrolysis ofp-nitrophenyl phosphate has been reported [51. It has been reported that Trp [6 and Arg residues [v are essential for activity and are situated at the active site of the enzyme. It has been found that the autocatalysis of N-phosphoamino acids could lead to some biochemical reactions in water/alcohol solution system such as the formation of N-phosphopeptides, N-phosphoryl amino acid esters, phosphorylester exchanged products 8 The present paper reported the effects of several N-thiophosphoryl amino acids on the activity of green crab alkaline phosphatase. The results suggest that N-thiophosphoryl-Cys and -Glu are non-competitive inhibitors, while N- thiophosphorymle, -Val, -Lys, -Ala and -Tyr are uncompetitive inhibitors. MATERIALS AND METHODS The alkaline phosphatase was prepared from green crab (Scylla Serrata) viscera first according to the method of Yan and Chen llu to the step of ammonium sulfate fractionation, the crude preparation was further chromatographed by ion-exchange with DEAE-cellulose, 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 protein. Synthesis and structural analysis of N-phosphoryl amino acids were as described by Lu et al. I121. p-nitrophenyl phosphate (pnpp) was from E. Merck. All other reagents were local products of analytical grade. The protein concentration was determined as described by Lowry [13], and the assay of green crab alkaline phosphatase was as described before [6]. Absorption and kinetic measurement were carried out on a Perkin-Elmer Lambda Bio spectrophotometer. Inhibitor studies were performed. The inhibitors were dissolved at the assay system containing p-nitrophenyl phosphate, 2 mm, MgC12, 2 mm and Na2CO3/NaHCO3 buffer, 50 mm (ph 10.0). 5 j.tl of the enzyme was added to l ml of the above assay system at 30 and the rate of substrate hydrolysis was monitored for 2 rain after a lag period of 20 sec. RESULTS Effects of the concentrations of several N-thiophosphoryl amino acids on the activity of green crab alkaline phosphatase The effects of the concentrations of various N-thiophosphoryl amino acids and respective free amino acids onpnpp hydrolysis of the enzyme were studied. Green crab alkaline phosphatase was inhibited by several N-phosphoryl amino acids as shown in Fig.1. With increasing the concentrations of all N-thiophosphoryl amino acids, the activity of green crab alkaline phosphatase 466
markedly decreased. Free cysteine, isoleucine and valine are inhibitors for alkaline phosphatase, while free glutamate, alanine, lysine and tyrosine have no effects for the activity of alkaline phosphotase. Compared with N-thiophsphoryl amino acids and respective free amino acids, it can be seen that N-phosphorylation of the amino acids led to increasing of inhibition ability for the enzyme activity except cysteine. The phosphorylation of cysteine siganifientlly decreased inhibition activity. Inhibition of N-thiophosphoryLCys and-glu following non-competitive mechanism The results illustrated in Fig. 2 show that double-reciprocal plots yield a family of straight lines with a common intercept on the 1/[S] axis but with different slopes, indicating that N- thiophosphoryl-cys is a competitive inhibitor. Secondary plot of the vertical intercept 1/Vm,vp versus concentration of N-thiophosphoryl-Cys is linear, as shown in the inset. The inhibition constant of N-thiophosphoryl-Cys obtained from the experimental data is recorded in Table 1. However, control experiment with free Cys shows that inhibition of alkaline phosphatase activity by Cys is mixed, as shown in Fig.3a. The equilibrium constant for inhibitor binding with free enzyme (E), K, can be obtained from the plot of the slopes versus the [Cys] (Fig. 3b), while equilibrium constant for inhibitor binding with enzyme-substrate complex (ES), Ks, can be obtained from the plot of the intercept versus the [Cys] (Fig.3c). The obtained constants are also summarized in Table 1. Similar results were obtained with N-thiophosphoryl-Glu. Secondary plot of the 1/Vm,pp versus concentration of N-thiophosphoryl-Glu is also a straight line. The inhibition constant of N- thiophosphoryl-glu obtained from the experimental data is also given in Table 1. The control experimental result with the free Glu shows no effect of the presence of the Glu on the enzyme activity. Inhibition of another 5 N-thiophosphoryl amino acids following au un-competitive mechanism Inhibition of the activity of green crab alkaline phosphatase by N-thiophosphoryl-Ile has been studied. Fig4 shows that the plots of 1/v versus 1/[S] give a family of parallel straight lines, indicating that N-thiophosphoryl-Ile is an un-competitive inhibitor, which binds at a site distinct from the substrate and binds only to the ES complex. Similarly, The Ks value can be obtained from the plot of 1/V,, app versus concentration of N-thiophosphoryl-Ile. the obtained result is also given in Table 1. 467
VOI. 45, No. 3, 1998 --u e,.q,,.= 0 e k eq cd D I J I I oo '. r (%) s (%) dl!a!*v L I e-4 O 7].= 0 (%) 2!A!V l J _ (%),(!t!a!lv r I 468
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 0.,- r :, "t Z" d (%) s o, 9 0 0.= '- (,,} (I!A!13 v '- (%) (]IA!13V I I o 469
0.6 4 "7.- 0.5 $ Ir 0.3 o. 0.2 0 I 2 g [EPS-Cys] mm _///s //f I I I z'/f/-t -2.0-1.0 0 I I I I 1.O 2.0 i l 3.0 1/[s] ( mm ) Fig.2. Lineweaver-Burk plots for inhibition of p-nitrophenylphosphate hydrolysis data by N- thiophosphoryl-cys. The experimental conditions were as for Fig. 1. The concentrations of N-thiophosphoryl-Cys for curves 0-4 were 0, 0.5, t.0, 1.5 and 2.0 ram, respectively. The inset represents the plot of apparent Michaelis-constant (Kin app) versus the concentration of N-thiophosphoryl-Cys to determine the inhibition constant. The line is drawn using linear least squares fit. Similar results were obtained with N-thiophosphoryl-Val and N-thiophosphoryl-Ala. The Ks values were also obtained from secondary plot. The above results show that the N- thiophosphoryl amino acids with hydrophobic side chain are un-competitive inhibitors for the enzyme. The control experimental results with free Ile and Val show that both the Ile and Val also are un-competitive inhibitors. However, the N-thiophosphorylation led to increasing of the their inhibition strength. No effect of free Ala on the enzyme activity was observed. It can be seen from the results in Fig. 1 that no effects of free Lys and Tyr on the activity of green crab alkaline phosphatase were observed. However, double-reciprocal plots show that both the N-thiophosphoryl-Lys and N-thiophosphoryl-Tyr are un-competitive inhibitors, the obtained inhibition constants are also recorded in Table 1. DISCUSSION It is well known that the alkaline phosphatase is an important enzyme participating in cell phospho-metabolism and that product HPO42 is a competitive inhibitors. It has been reported that some amino acids and substrate analogues have inhibition effects on the activity of alkaline phosphatase I14'15]. However, the effects of the derivatives of amino acids on alkaline phospbatase 470
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 2.0 4 0.75 1.5 0.25./j. 9 3 1 2 3 4 1.0 / 2 [C 3 s] (nlm) 10 O.3 0.5 '0.2 0.1 * I! I I -I.0 0 1.0 2.0 0 1 2 3 4 1/[s] (ram) 4 [Cys] (ram) Fig.3. Inhibition ofp-nitrophenylphosphate hydrolysis by L-cysteine. The experimental conditions were as for Fig.l except that inhibitor was L-cysteine. (a) Lineweaver-Burk plots ofpnpp hydrolysis. Concentration of L-cysteine for curves 0-4 were 0, 1.0, 2.0, 3.0 and 4.0 mm, respectively. (b) Secondary plot of the slopes of the straight lines versus L- cysteine concentration. (c) Secondary plot of the intercept of the straight lines versus L-cysteine concentration. The line is drawn using linear least squares fit. Table 1. Inhibition types and inhibition constants of the activity of green crab alkaline phosphatase by N-thiophosphoryl amino acids and respective free amino acids. Inhibitor Inhibition type Inhibition constant (ram) KI (ram) Kis (ram) L-cysteine Mixed* 1.50 5.70 N-thiophosphoryl-Cys non-competitive 4.45 4.45 L-glutimate no inhibition - N-thiophosphoryl-Glu non-competitive 5.66 5.66 L-isoleucine un-competitive 5.87 N-thiophosphoryMle un-competitive 2.94 L-valine un-competitive 7.20 N-thiophosphoryl-Val un-competitive 5.26 L-alanine no inhibition - N-thiophosphoryl-Ala un-competitive 8.34 L-lysine no inhibition' N-thiophosphoryl-Lys un-competitive 6.06 L-tyrosine no inhibition - - N-thiophosphoryl-Tyr un-competitive 11.74 * Mixed un-competitive and non-petitive inhibition. 471
u 45, No. 3, 1998,ol,0.4 0.8 4 0 L I " 0 2 4../2. [EPS-11e],riM"." /.,---/I"/'. 0.2 i ' /.I /.-'J -2.0-1.0 0, I a I J I 1.0 2.0 3.0 1/[sJ ( mm ) Fig.4. Lineweaver-Burk plots for inhibition of p-nitrophenylphosphate hydrolysis by N- thiophosphoryl-ile. The experimental conditions were as for Fig. 1 except that the inhibitor was N- thiophosphorymle. Concentrations of N-thiophosphoryMle for curves 0-4 were 0, 1.0, 2.0, 3.0 and 4.0 ram, respectively. The inset represents the plot of l/vmax versus N-thiophosphoryl-Ile concentration. The line is drawn using linear least squares fit. activity has been little explored. In the present paper, inhibitions of the enzyme activity by N- thiopbosphoryl amino acids were investigated, the obtained results show that among several N- thiophosphoryl amino acids in the present investigation only N-thiophosphoryl-Cys and N- thiophosphoryl-glu are non-competitive inhibitors, another are un-competitive inhibitors, the inhibition effects of all N-thiophosphoryl amino acids on the enzyme actwity are strongger than that of respective free amino acids except the N-thiophosphoryl-Cys. It is known that inhibition effect of free Cys contained both acting mechanisms of reversible inhibition and reduction of the essential disulfide bond. It is possible N-thiophosphorylation greatly decreased the reduction ability, and led to that inhibition of N-thiophosphoryl-Cys was weaker than free Cys. In addition, results also show that mixed type for free Cys was changed into non-competitive inhibition for N-thiophosphoryl- Cys. For the un-competitive inhibitors, the inhibition strength follows the order N-thiophosphoryl- Ile > -Val > -Lys > -Ala > -Tyr. It can be seen that among these un-competitive inhibitors three have the hydrophobic side chains, and the side chain is larger, the inhibition is strongger. The results also show that the inhibition strength of N-thiophosphoryl amino acids is strongger than that of respective free amino acids except the N-thiophosphoryl-Cys. It can be suggested that N- phosphorylation of amino acids increased their inhibition strength. 472
ACKNOWLEDGMENTS The present investigation was supported in part by Grant 39470561 of the China Natural Science Foundation and by Grant from SECD Laboratory of Life Organic Sulforus for Q.X. Chen. We wish to give thanks to prof. Y.F. Zhao for her guidance in the present investigation. REFERENCES 1. Fernley, H. N. (1971) The Enzymes (Edited by Boyer, P. D.), 3rd edn. Vol. IV, pp. 417-447. 2. McComb, R. B., Bowers, G. N. and Posen, S. (1979) 3, Coleman, J. E. (1992) A. Rev. Biophys. Biomol. Struct. 24, 441-483. 4. Kim, E.E. and Wyckoff, H. W. (1991) J. Mol. Biol. 281,449. 5. Chen, Q X., Zhang, W. Wang, H. R. and Zhou, H. M. (1996) Int. J. Biol. Macromoll 19, 257-261. 6. Chen, Q. x., Zhang, W., Zheng, W. Z., Zhao, H., Yan, S. X., Wang, H. R. and Zhou, H. M. (1996) J. Protein Chem. 15, 345-350. 7. Xie, W.Z., Wang, H.R., Chen, Q.X. and Zhou, H, M. (1996) Biochem. Mol. Biol. Int. 40(5), 981-991. 8. Li, Y. M., Yin, Y. W. and Zhao, Y. F. (1992) Int. J. Peptide Protein Res. 39, 375-381 9. Ma, X. B. and Zhao, Y. F. (1992) Phosphorus, Sulfur and Silicon 66, 107-114 10. Li, Y. C., Tan, B. and Zhao, Y. F. (1993) Heteratom Chemistry 4, 415-419 11. Yan, S. X. and Chen, Q. X (1985)J. Xiamen University (in Chinese)24, 367-372. 12. Lu, H.Y., Zhang, N.J. and Zhao, Y.F. (1997) Synth. Commun. (in press). 13. Lowry, O. H. (1951)J. Biol. Chem_ 193, 265-275. 14. Cyboron, G.W. and Wuthier, R.E. (1981) J. Biol. Chem. 256, 7262-7268. 15. Asgeirsson, B., Hartemink, R. and Chlebowski, J.F. (1995) Comp. Biochem. Physiol. l l0b, 315-329. 473