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Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site
TSINGHUA SCIENCE AND TECHNOLOGY ISSN 1007-0214 02/20 pp414-420 Volume 10, Number 4, August 2005
Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site* CHEN Hongtao (ч܀ඁ)1, XIE Liping (ऽ)1,2, YU Zhenyan (ဟჲཧ)1, ZHANG Rongqing (ఔ)1,2,** 1. Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; 2. Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China Abstract: Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde (OPA). The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 (mmol/L)–1·min–1 at pH 7.5 and 25°C. A Tsou’s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme. Key words: alkaline phosphatase; Pinctada fucata; chemical modification; kinetics
Introduction Alkaline phosphatase (ALP, EC 3.1.3.1) is a substrate nonspecific phosphomonoesterase that catalyzes hydrolysis of a wide variety of phosphomonoesters under alkaline conditions. In the presence of a phosphate acceptor such as Tris or ethanolamine, ALP also acts as a transphosphorylase[1]. A detailed catalytic mechanism of the enzyme has been proposed, based on kinetic and structural studies of the native and a number of site-directed mutant Escherichia coli Received: 2004-05-14 γ Supported by the National High-Tech Research and Development (863) Program of China (No. 2003AA603430)
γγ To whom correspondence should be addressed. E-mail: [email protected]; Tel: 86-10-62772899
(E. coli) ALP. The reaction follows ping-pong replacement kinetics and proceeds through a phosphoseryl intermediate to yield inorganic phosphate or to transfer the phosphoryl group to another alcohol[2,3]. Although its detailed physiological functions have not been fully elucidated, the wide distribution of ALP in almost all living beings indicates its essential role in cell metabolism. In marine organisms, ALP is believed to be involved in the absorption of phosphate and calcium from seawater and the biomineralization process. The ALP activity has been used as a stress marker to evaluate the effects of environmental exotics, such as heavy metals and organic chlorides, on marine organisms[4,5]. The crystal structures of ALP from E. coli, Pandalus borealis and human placenta have been solved[1,6,7]. They are all homodimers with very similar overall topologies and their active site architectures all had a
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
metal triad (usually two zinc ions and one magnesium ion). Comparative studies of the primary sequence of ALPs from E. coli, Saccharomyces cerevisiae, P. borealis, chicken, and humans showed 25%-45% sequence identity among ALPs from different species[8]. Most of the important functional residues, including the serine nucleophile (Ser-112 in E. coli ALP) and an arginine residue (Arg-166 in E. coli ALP) that is involved in substrate binding, are highly conserved, which implies that similar catalytic mechanisms are shared among ALPs from different species. Although ALPs from bacteria and mammals have been extensively studied, there are few reports about ALPs from mollusca, the second largest phylum of the animal kingdom. Pinctada fucata (P. fucata), which belongs to the family Pteriidae of bivalve mollusca, is a key source of marine pearl production. ALP is believed to be involved in phosphate and calcium absorption in oysters and in the biomineralization process. A better understanding of oyster ALP would help identify the mechanism of shell and pearl formation. A previous paper illustrated the enzymatic properties of the tissue-nonspecific ALP from P. fucata[9]. This paper reports on the OPA modification of this ALP.
1
Materials and Methods
1.1
Animal and chemicals
Adult P. fucata were harvested from the Beihai Oyster Culture Centre, Guangxi Zhuang Autonomous Region, China. p-Nitrophenyl phosphate (pNPP) and o-phthalaldehyde (OPA) were from Amresco. 2,4,6Ttrinitrobenzene-sulfonic acid (TNBS), diethylpyrocarbonate (DEPC), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and L-histidyldiazobenzylphosphonic acid agarose were Sigma products. All other chemicals were local products of analytical grade. 1.2
Enzyme preparation and activity assays
The ALP from P. fucata was prepared as previously described[9] to the step of Sephadex G-150 gel filtration. The active fractions were collected and evaluated using an affinity chromatography as described below. The active peaks were then combined and dialyzed for 72 h to remove the inorganic phosphate. All the purification procedures were carried
415
out at 4ć. The final preparations, with specific activity of 1215 unitgmg–1, showed homogeneity on polyacrylamide gel electrophoresis in both the absence and presence of SDS. The standard enzyme activity assay was carried out as previously described[9]. The released p-nitrophenol was determined spectrometrically by measuring the absorbance increase at 405 nm using a molar extinction coefficient of 17.3 (mmol/L)–1gcm–1. One unit of activity represents the amount of enzyme required to produce 1 µmol product per minute under the assay conditions. 1.3
Inactivation of ALP from P. fucata by OPA
All the reactions were carried out at (25±1)ć. The Tris that would interfere with the reaction was removed by first passing the enzyme through a Sephadex G-50 column previously equilibrated with 20 mmol/L Hepes buffer (pH 7.5). The OPA solutions were freshly made in 1% methanol. The modification procedure was carried out by incubating ALP with different concentrations of OPA in 50 mmol/L bicarbonate buffer, pH 9.0. The final concentration of the enzyme was 2.2 µmol/L. A control tube was maintained with the same amount of enzyme, but without any OPA. At various time intervals, aliquots were withdrawn and the reaction was terminated by the addition of an equal volume of stopping solution (5 mmol/L cysteine and 5 mmol/L 2-mercaptoethanol). The reaction mixture was ultra-centrifuged with a Centricon (millipore) apparatus to remove the excess OPA and then diluted 10 folds by 20 mmol/L Tris-HCl buffer. The residual enzyme activity was then assayed. 1.4
Stoichiometry of OPA inactivation
Various partially modified enzymes were obtained by mixing 2.3 µmol/L ALP with OPA at specific molar proportions ranging from 1:20 to 1:1000. After a period of time, the number of formed isoindole derivatives was determined from the absorbance increase at 337 nm using a molar extinction coefficient of 7.66 (mmol/L)–1gcm–1 (Ref. [10]). The mixture was then ultra-centrifuged, diluted, and assayed to determine the residual activity. 1.5
Protection experiments
The enzyme was preincubated with varying concen-
416
trations of glycerol 3-phosphate, AMP, phosphate or tungstate, at 25ć in 50 mmol/L sodium bicarbonate buffer (pH 9.0) for 5 min. 1 mmol/L OPA was then added. After 30 min, the reaction was terminated and the residual activity was measured after ultracentrifugation and dilution. Control experiments without OPA were run concurrently. 1.6
Double modification studies
The enzyme (2.3 µmol/L) was preincubated with 0.2 mmol/L TNBS (in 50 mmol/L bicarbonate buffer, pH 9.0), 1.0 mmol/L DTNB (in 20 mmol/L Hepes buffer, pH 8.5), or 2.0 mmol/L DEPC (in 20 mmol/L Hepes buffer, pH 7.0) for 2 h at (4±1)ćfor complete modification. The modified samples were then treated with 1.0 mmol/L OPA for 1 h. The formation of isoindole derivatives was monitored by recording the fluorescence emission spectra excited at 337 nm. 1.7
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
2
Results
2.1
Inhibition of ALP from P. fucata by OPA
Incubation of ALP from P. fucata with increasing concentrations of OPA resulted in a progressive decrease of the enzyme activity. The activity loss could not be restored by dialysis or ultra-centrifugation, which indicated an irreversible reaction. Figure 1 shows that the inactivation follows pseudo first-order kinetics. The second-order rate constant was calculated by replotting the pseudo first-order rate constants (kobs) against OPA concentration. A linear relationship was observed and the second-order rate constant of 0.167 (mmol/L)–1gmin–1 was determined from the slope (Fig. 1 inset). Analysis using Lery’s method of plotting log kobs against the logarithm of the OPA concentration yielded a slope of 0.89 (Fig. 2), which indicated that at least one mole of OPA per mole of active site was required to inactivate the enzyme[11].
Affinity chromatography experiments
The native or OPA-modified enzymes were loaded onto an L-histidyldiazobenzylphosphonic acid agarose column ( 1.0 cm×2.5 cm) pre-equilibrated with 20 mmol/L Tris-HCl buffer, pH 7.5. The enzymes were eluted with the same buffer containing 10 mmol/L KH2PO4 at a speed of 1 mLgmin–1. The absorbence at 280 nm of the eluted enzyme was then monitored. 1.8
CD spectra measurements
The far-ultraviolet circular dichroism (CD) spectra were recorded on a Jasco-J715 spectropolarimeter using a 2-mm path length cell. The recorded spectra were averages of four scans ranging from 195 to 250 nm to ensure a good signal-to-noise ratio. All spectra were recorded in 20 mmol/L Tris-HCl buffer, pH 7.5, at 25ć, with final enzyme concentrations of 2.2 µmol/L. 1.9
Fig. 1 Kinetics of inactivation of ALP from P. fucata by OPA. The OPA concentration for lines 1-5 was 50, 100, 150, 200, and 250 Pmol/L, respectively. The inset shows the second-order plot of the pseudo-first rate constants as a function of log OPA concentration.
Other methods
Protein concentrations were measured with a BCA protein assay reagent (BCA kit, Pierce) using bovine serum albumin as the standard. The molecular mass of the dimer enzyme was estimated to be 86 kD from gel filtration experiments. Fig. 2 Lery’s plot of OPA inactivation. The slope gives a reaction order of 0.89.
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
417
The far-UV CD spectra of both native and fully modified enzyme samples were recorded with almost identical spectra observed (Fig. 3), suggesting that modification does not result in gross changes of the enzyme conformation.
residues is the value of i which gives a straight line when plotting Ax/A0, the fraction of the fully active enzyme, against the number of isoindoles modified (m). The results in Fig. 4 show that the ALP activity is dependent upon formation of only one isoindole group.
Fig. 3 CD spectra of native and OPA-modified ALP from P. fucata. Far-UV spectra were recorded for native (ƹ) and OPA-modified (Ƹ ) enzyme at 25ć. Each spectrum represents the average of four independent scans.
Fig. 4 Correlation between the number of isoindole formed and the residual activity of ALP from P. fucata. Enzyme with a concentration 2.3 µmol/L was incubated with OPA at molar proportions from 1:20 to 1:1000. After incubation, the residual activity and the number of isoindoles formed were measured as described in the text. The data is presented as a Tsou’s plot for i = 1 (Ŷ), i = 2 (ż), and i = 3 (Ÿ).
2.2
Stoichiometry of OPA inactivation
The inactivated enzyme exhibited a new absorbance maximum at 337 nm, which is characteristic of isoindole absorption. An emission peak was also observed at 415 nm in the fluorescence emission spectrum excited at 337 nm. These results indicated that inactivation of the enzyme by OPA resulted in the formation of isoindole groups from the reaction of OPA and the enzyme[12]. The kinetic data showed that at most one isoindole derivative was produced per mole by the inactivation (1 mole isoindole was derived from 1 mole OPA). The limitations of kinetic methods in determining the number of modifiable sites essential for enzyme activity were described by Lery[11]. Therefore, the stoichiometry of the OPA inactivation was studied by quantitatively monitoring the isoindole formation with a Tsou’s plot analysis used to determine the number of isoindoles essential for the activity[13]. Assuming that the reactivities of all n reactive sites toward OPA are approximately equal and that modification of any one would inactivate the enzyme, the relationship between the residual activity, Ax, against residue modification will be ( Ax / A0 )1/ i
( n m) / n
(1)
where A0 is the initial activity. The number of essential
2.3
Double modification
The critical residues related to the formation of isoindole derivatives was investigated using double modification experiments. The enzyme was completely inactivated after incubation with 2.0 mmol/L DEPC (a regent for histidine modification) and lost 80% of the initial activity after pretreatment by 0.2 mmol/L TNBS (a regent for lysine modification). However, the enzyme retained 90% of its initial activity after incubation with 1.0 mmol/L DTNB for 2 h. Remodification of the DTNB (a regent for cysteine modification) modified enzyme by 1.0 mmol/L OPA caused complete inactivation. The native enzyme, enzymes modified by TNBS, DEPC, or DTNB did not present an emission peak at 415 nm in the fluorescence spectrum excited at 337 nm (data not shown), and therefore indicated no formation of isoindole derivatives. Figure 5 shows the effects of these residual specific modifiers on the isoindole fluorescence of the OPA modified enzyme. Complete premodification by DEPC or TNBS eliminated the characteristic isoindole derivative peak at 415 nm. However, pretreatment by DTNB did not prevent the formation of isoindole
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
418
fluorescence. These results strongly indicate that no cysteine residue was involved in the isoindole formation during the OPA inactivation. Thus, the inactivation was due to formation of an isoindole derivative involving lysine and histidine residues.
2.5
Affinity chromatography experiments
The substrate binding ability of the OPA-treated enzyme was evaluated on an ALP specific affinity column. Experiments were also conducted using the same amount of native enzyme. The elution graphs are shown in Fig. 6. The OPA-modified enzyme lost the ability to bind in the column and thus was directly washed out. However, the native enzyme could be captured by the column and then eluted with the competitive phosphate, which indicated the specific binding ability.
Fig. 5 Effect of lysine, histidine, and cysteine modifiers on the isoindole fluorescent of ALP from P. fucata. The enzyme (2.3 µmol/L) was incubated with 2.0 mmol/L DEPC, 0.2 mmol/L TNBS, or 1.0 mmol/L DTNB at 25°C for complete modification, respectively. The modified enzyme samples were then treated with 1.0 mmol/L OPA for 1 h. The change in the isoindole fluorescence was monitored at an excitation wavelength of 337 nm.
2.4
Protection experiments
The substrates 2-glycerol phosphate and adenosine monophosphate (AMP) were found to significantly protect the enzyme from inactivation by OPA. About 60% of the activity could be retained by incubating with 5 mmol/L 2-glycerol phosphate or 10 mmol/L AMP. Phosphate and tungstate, which are powerful competitive inhibitors, also markedly protected the enzyme against inactivation (Table 1). Table 1
Fig. 6 Affinity chromatography experiments for the modified ALP from P. fucata. The OPA-modified enzyme (a) and the native enzyme (b) were loaded onto an L-histidyldiazobenzylphosphonic acid agarose column which is specific to binding ALPs. An elution speed of 1 mLgmin–1 was applied and A280 was monitored for the eluted enzyme.
Inhibitors against OPA inactivation Residual Incubation system
activity (%)
Enzyme
100
+ OPA (200 µmol/L)
11
+ ȕ-GP (5 mmol/L) + OPA (200 µmol/L)
70
+ AMP (5 mmol/L) + OPA (200 µmol/L)
65
+ tungstate (10 mmol/L) + OPA (200 µmol/L)
61
+ phosphate (10 mmol/L) + OPA (200 µmol/L)
63
The values given are means of three independent sets of experiments with standard deviation less than 10%.
3
Discussion
OPA is a bifunctional cross-linking reagent that has been extensively used to detect the active site of a number of enzymes[14,15]. However, the modification of alkaline phosphatase, which is widely distributed in most living beings, by this reagent has not been widely investigated. The present studies showed that ALP from P. fucata could be irreversibly inactivated by OPA. The inactivation followed pseudo-first-order kinetics with the second-order inhibition rate constant
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
calculated to be 0.167 (mmol/L)–1gmin–1. The CD spectra of both native and modified enzymes were almost identical, indicating that the inhibition does not result in gross changes in the enzyme conformation and that inactivation was due to modification of essential residues at the active site. A combination of kinetic and statistical methods shows that the OPA inactivation is dependent upon formation of only one isoindole group. OPA is generally known to form an isoindole derivative by cross-linking thiol and amino groups[16]. However, DTNB titration of the OPA-modified enzymes showed no change in the number of thiol groups (data not shown). Furthermore, ALP from P. fucata was not inhibited by thiol reagents such as DTNB, indicating that thiol groups are not involved in the enzyme activity, which is consistent with ALPs from other sources[1,6,7]. Fluorescence studies indicated that modification of either lysine residue by TNBS or histidine residues by DEPC prevented formation of the isoindole derivative. Thus, these results prove the involvement of both lysine and histidine in the formation of the isoindole derivative. A proposed mechanism for the reaction of OPA with histidine and lysine residues[17] suggests that the aldehyde groups of OPA react with the secondary amine in the imidozole ring and the İ-amine in the lysine residue to form the fluorescent isoindole derivative. The importance of histidine residues in the catalytic activity of ALPs has been shown in many investigations. Histidines serve as ligands to the metals at the active site and are highly conserved in the active site of ALP from E. coli to humans[1,6,7]. Lysine residues are negatively charged and are potentially involved in anionic substrate binding[18,19]. In E. coli ALP, a lysine residue forms a secondary interaction to the substrate through a water molecule[20]. In Scylla serrata, a lysine residue is essential for the enzyme activity[21]. The inactivation of ALP from P. fucata by OPA strongly suggests the existence of histidine and lysine residues at or near the active site of this molluscan ALP. Protection experiments were performed to determine the effect of substrates and competitive inhibitors on OPA-induced inactivation. The results showed that 2-glycerol phosphate and AMP effectively protected the enzyme from inactivation, as well as the competitive inhibitors, phosphate, and tungstate. The
419
binding experiments confirmed that after the OPA modification, the enzyme lost the ability to bind with the specific affinity column. These results indicate that substrate binding can markedly affect the accessibility of OPA and the OPA modification can effectively prevent binding of the substrate, which leaves little doubt that the isoindole group formed by OPA inhibition is located at the substrate binding site of the ALP from P. fucata. The isoindole derivative formed in the enzyme active site could be used as an indicator to detect the polarity of the micro-environment of the active site[22]. There are many successful applications of OPA as a probe to ascertain the conformational flexibility of the active site[23,24]. Our studies demonstrated the formation of a fluorescent chemoaffinity label at the active site by OPA modification of ALP which can be used as a powerful tool to study the detailed structure-function relationship of the ALP of P. fucata to contribute to the study of the mechanism of marine biomineralization. Acknowledgements The author thanks Li Yu and Jia Wei for their enthusiastic help with the enzyme purification and Dr. Ma Zhuojun for his help with the oyster culture.
References [1] Kim E E, Wyckoff H W. Reaction mechanism of alkaline phosphatase based on crystal structures: Two-metal ions catalysis. J. Mol. Biol., 1991, 218: 449-464. [2] Stec B, Holtz K M, Kantrowitz E R. A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J. Mol. Biol., 2000, 299: 1303-1311. [3] Holtz K M, Kantrowitz E R. The mechanism of alkaline phosphatase reaction: Insights from NMR, crystallography and site-specific mutagenesis. FEBS Letter, 1999, 462: 7-11. [4] Mazorra M T, Bubio J A, Blasco J. Acid and alkaline phosphatase activities in the clam Scrobicularia plana: Kinetic characteristics and effect of heavy metals. Comp. Biochem. Phys. B, 2002, 131: 241-249. [5] Reddy M S, Rao K V. Methylparathion-induced alterations in the acethylcholinesterase and phosphatase in a penaeid prawn, Metapenaeus monoceros. Bull. Environ. Contam. Toxicol., 1990, 45: 350-357. [6] de Bacher M, McSweeney S, Rasmussen H B, Riise B W,
420
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
Lindley P, Hough E. The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase. J. Mol. Biol., 2002, 318: 1265-1274.
[16] Quang K H. Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate
synthase
by
o-phthalaldehyde. J. Biol. Chem., 1990, 265: 6700-6704.
[7] le Du M H, Stigbrand T, Taussig M J, Menez A, Stura E A.
[17] George S P, Rao M B. Conformation and polarity of the
Crystal structure of alkaline phosphatase from human
active site of xylanase I from Thernomonospora sp. as
placenta at 1.8 Å resolution: Implication for a substrate
deduced by fluorescent chemoaffinity labeling. Eur. J.
specificity. J. Mol. Biol., 2001, 276: 9158-9165.
Biochem., 2001, 268: 2881-2888.
[8] Nilsen I W, Øverbø K, Olsen R L. Thermolabile alkaline
[18] Gite S, Reddy G, Shankar V. Active site characterization
phosphatase from Northern shrimp (Pandalus borealis):
of S1 nuclease: 1. Affinity purification and influence of
Protein and cDNA sequence analyses. Comp. Biochem.
amino-group modification. Biochem. J., 1992, 285:
Phys. B, 2001, 129: 853-861. [9] Xiao R, Xie L P, Lin J Y, Li C H, Chen Q X, Zhou H M,
489-494. [19] Richardson R M, Parés X, Cuchillo C M. Chemical
Zhang R Q. Purification and enzymatic characterization of
modification
alkaline phosphatase from Pinctada fucata. J. Mol. Catal.
cyclohexane-1,2-dione indicates the Lys-7 and Arg-10 are
B Enzym., 2002, 17: 65-74.
involved in the P2 phosphate-binding subsite of bovine
[10] Simons S S Jr, Johnson D F. Reaction of o-phthalaldehyde and thiol with primary amines: Formation of 1-alkyl (and aryl) thiol-2-alkylisoindoles. J. Org. Chem., 1978, 43: 7769-7775. [11] Lery H M, Leber P A, Ryan I M. Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphate and other phosphate compounds. J. Biol. Chem., 1963, 238: 3654-3659. [12] Harris G W, Jenkins J A, Connerton I, Pickersgill R W. Defined crystal structure of the catalytic domain of xylanase A from Pseudomonos fluorecens at 1.8 Å resolution. Acta Cryst., 1996, D52: 393-401.
by
pyridoxal
5’-phosphate
and
pancreatic ribonuclease A. Biochem. J., 1990, 267: 593-599. [20] Sun L, Carolan D C, Kantrowitz E R. Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center. Biochemistry, 1999, 38: 2842-2848. [21] Chen Q X, Zhou H M. An essential lysine residue of green crab (Scylla serrata) alkaline phophatase. Biochem. Mol. Bio. Int., 1998, 46: 225-231. [22] Palczewski
K,
Hargrave
P
A,
Kochman
M.
o-Phthalaldehyde, a fluorescence probe of aldolase active site. Eur. J. Biochem., 1983, 137: 429-435.
[13] Tsou C L. Relation between modification of functional
[23] Wang Z F, Xu Y K, Zhou H M. Kinetics of irreversible
groups of proteins and their biological activity: 1. A
inhibition of creatine kinase during modification by
graphical method for the determination of the number and
o-phthaldehyde. Enzyme Protein, 1995, 48: 1-9.
type of essential groups. Sci. Sin., 1962, 11: 1535-1558.
[24] Heng Y B, Wang Z, Chen B Y, Wang X C. Multiple
[14] Anjali P, Saifuddin S, Sarvagya K S. Identification of
effects of chemical reagent on enzyme: o-Phthalaldehyde-
cysteine and lysine residues present at the active site of
induced inactivation, dissociation and partial unfolding of
beef liver glutamate dehydrogenase by o-phthalaldehyde.
lactate dehydrogenase from pig heart. Int. J. Bio.
Biochim. Biophys. Acta B: Pro. Struc. Mol. Enzymol., 1996,
Macromol., 2003, 32: 191-197.
1293: 122-128. [15] Ahn J Y, Choi S, Cho S W. Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde. Biochimie, 1999, 81: 1123-1129.
Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site* CHEN Hongtao (ч܀ඁ)1, XIE Liping (ऽ)1,2, YU Zhenyan (ဟჲཧ)1, ZHANG Rongqing (ఔ)1,2,** 1. Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; 2. Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China Abstract: Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde (OPA). The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 (mmol/L)–1·min–1 at pH 7.5 and 25°C. A Tsou’s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme. Key words: alkaline phosphatase; Pinctada fucata; chemical modification; kinetics
Introduction Alkaline phosphatase (ALP, EC 3.1.3.1) is a substrate nonspecific phosphomonoesterase that catalyzes hydrolysis of a wide variety of phosphomonoesters under alkaline conditions. In the presence of a phosphate acceptor such as Tris or ethanolamine, ALP also acts as a transphosphorylase[1]. A detailed catalytic mechanism of the enzyme has been proposed, based on kinetic and structural studies of the native and a number of site-directed mutant Escherichia coli Received: 2004-05-14 γ Supported by the National High-Tech Research and Development (863) Program of China (No. 2003AA603430)
γγ To whom correspondence should be addressed. E-mail: [email protected]; Tel: 86-10-62772899
(E. coli) ALP. The reaction follows ping-pong replacement kinetics and proceeds through a phosphoseryl intermediate to yield inorganic phosphate or to transfer the phosphoryl group to another alcohol[2,3]. Although its detailed physiological functions have not been fully elucidated, the wide distribution of ALP in almost all living beings indicates its essential role in cell metabolism. In marine organisms, ALP is believed to be involved in the absorption of phosphate and calcium from seawater and the biomineralization process. The ALP activity has been used as a stress marker to evaluate the effects of environmental exotics, such as heavy metals and organic chlorides, on marine organisms[4,5]. The crystal structures of ALP from E. coli, Pandalus borealis and human placenta have been solved[1,6,7]. They are all homodimers with very similar overall topologies and their active site architectures all had a
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
metal triad (usually two zinc ions and one magnesium ion). Comparative studies of the primary sequence of ALPs from E. coli, Saccharomyces cerevisiae, P. borealis, chicken, and humans showed 25%-45% sequence identity among ALPs from different species[8]. Most of the important functional residues, including the serine nucleophile (Ser-112 in E. coli ALP) and an arginine residue (Arg-166 in E. coli ALP) that is involved in substrate binding, are highly conserved, which implies that similar catalytic mechanisms are shared among ALPs from different species. Although ALPs from bacteria and mammals have been extensively studied, there are few reports about ALPs from mollusca, the second largest phylum of the animal kingdom. Pinctada fucata (P. fucata), which belongs to the family Pteriidae of bivalve mollusca, is a key source of marine pearl production. ALP is believed to be involved in phosphate and calcium absorption in oysters and in the biomineralization process. A better understanding of oyster ALP would help identify the mechanism of shell and pearl formation. A previous paper illustrated the enzymatic properties of the tissue-nonspecific ALP from P. fucata[9]. This paper reports on the OPA modification of this ALP.
1
Materials and Methods
1.1
Animal and chemicals
Adult P. fucata were harvested from the Beihai Oyster Culture Centre, Guangxi Zhuang Autonomous Region, China. p-Nitrophenyl phosphate (pNPP) and o-phthalaldehyde (OPA) were from Amresco. 2,4,6Ttrinitrobenzene-sulfonic acid (TNBS), diethylpyrocarbonate (DEPC), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and L-histidyldiazobenzylphosphonic acid agarose were Sigma products. All other chemicals were local products of analytical grade. 1.2
Enzyme preparation and activity assays
The ALP from P. fucata was prepared as previously described[9] to the step of Sephadex G-150 gel filtration. The active fractions were collected and evaluated using an affinity chromatography as described below. The active peaks were then combined and dialyzed for 72 h to remove the inorganic phosphate. All the purification procedures were carried
415
out at 4ć. The final preparations, with specific activity of 1215 unitgmg–1, showed homogeneity on polyacrylamide gel electrophoresis in both the absence and presence of SDS. The standard enzyme activity assay was carried out as previously described[9]. The released p-nitrophenol was determined spectrometrically by measuring the absorbance increase at 405 nm using a molar extinction coefficient of 17.3 (mmol/L)–1gcm–1. One unit of activity represents the amount of enzyme required to produce 1 µmol product per minute under the assay conditions. 1.3
Inactivation of ALP from P. fucata by OPA
All the reactions were carried out at (25±1)ć. The Tris that would interfere with the reaction was removed by first passing the enzyme through a Sephadex G-50 column previously equilibrated with 20 mmol/L Hepes buffer (pH 7.5). The OPA solutions were freshly made in 1% methanol. The modification procedure was carried out by incubating ALP with different concentrations of OPA in 50 mmol/L bicarbonate buffer, pH 9.0. The final concentration of the enzyme was 2.2 µmol/L. A control tube was maintained with the same amount of enzyme, but without any OPA. At various time intervals, aliquots were withdrawn and the reaction was terminated by the addition of an equal volume of stopping solution (5 mmol/L cysteine and 5 mmol/L 2-mercaptoethanol). The reaction mixture was ultra-centrifuged with a Centricon (millipore) apparatus to remove the excess OPA and then diluted 10 folds by 20 mmol/L Tris-HCl buffer. The residual enzyme activity was then assayed. 1.4
Stoichiometry of OPA inactivation
Various partially modified enzymes were obtained by mixing 2.3 µmol/L ALP with OPA at specific molar proportions ranging from 1:20 to 1:1000. After a period of time, the number of formed isoindole derivatives was determined from the absorbance increase at 337 nm using a molar extinction coefficient of 7.66 (mmol/L)–1gcm–1 (Ref. [10]). The mixture was then ultra-centrifuged, diluted, and assayed to determine the residual activity. 1.5
Protection experiments
The enzyme was preincubated with varying concen-
416
trations of glycerol 3-phosphate, AMP, phosphate or tungstate, at 25ć in 50 mmol/L sodium bicarbonate buffer (pH 9.0) for 5 min. 1 mmol/L OPA was then added. After 30 min, the reaction was terminated and the residual activity was measured after ultracentrifugation and dilution. Control experiments without OPA were run concurrently. 1.6
Double modification studies
The enzyme (2.3 µmol/L) was preincubated with 0.2 mmol/L TNBS (in 50 mmol/L bicarbonate buffer, pH 9.0), 1.0 mmol/L DTNB (in 20 mmol/L Hepes buffer, pH 8.5), or 2.0 mmol/L DEPC (in 20 mmol/L Hepes buffer, pH 7.0) for 2 h at (4±1)ćfor complete modification. The modified samples were then treated with 1.0 mmol/L OPA for 1 h. The formation of isoindole derivatives was monitored by recording the fluorescence emission spectra excited at 337 nm. 1.7
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
2
Results
2.1
Inhibition of ALP from P. fucata by OPA
Incubation of ALP from P. fucata with increasing concentrations of OPA resulted in a progressive decrease of the enzyme activity. The activity loss could not be restored by dialysis or ultra-centrifugation, which indicated an irreversible reaction. Figure 1 shows that the inactivation follows pseudo first-order kinetics. The second-order rate constant was calculated by replotting the pseudo first-order rate constants (kobs) against OPA concentration. A linear relationship was observed and the second-order rate constant of 0.167 (mmol/L)–1gmin–1 was determined from the slope (Fig. 1 inset). Analysis using Lery’s method of plotting log kobs against the logarithm of the OPA concentration yielded a slope of 0.89 (Fig. 2), which indicated that at least one mole of OPA per mole of active site was required to inactivate the enzyme[11].
Affinity chromatography experiments
The native or OPA-modified enzymes were loaded onto an L-histidyldiazobenzylphosphonic acid agarose column ( 1.0 cm×2.5 cm) pre-equilibrated with 20 mmol/L Tris-HCl buffer, pH 7.5. The enzymes were eluted with the same buffer containing 10 mmol/L KH2PO4 at a speed of 1 mLgmin–1. The absorbence at 280 nm of the eluted enzyme was then monitored. 1.8
CD spectra measurements
The far-ultraviolet circular dichroism (CD) spectra were recorded on a Jasco-J715 spectropolarimeter using a 2-mm path length cell. The recorded spectra were averages of four scans ranging from 195 to 250 nm to ensure a good signal-to-noise ratio. All spectra were recorded in 20 mmol/L Tris-HCl buffer, pH 7.5, at 25ć, with final enzyme concentrations of 2.2 µmol/L. 1.9
Fig. 1 Kinetics of inactivation of ALP from P. fucata by OPA. The OPA concentration for lines 1-5 was 50, 100, 150, 200, and 250 Pmol/L, respectively. The inset shows the second-order plot of the pseudo-first rate constants as a function of log OPA concentration.
Other methods
Protein concentrations were measured with a BCA protein assay reagent (BCA kit, Pierce) using bovine serum albumin as the standard. The molecular mass of the dimer enzyme was estimated to be 86 kD from gel filtration experiments. Fig. 2 Lery’s plot of OPA inactivation. The slope gives a reaction order of 0.89.
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
417
The far-UV CD spectra of both native and fully modified enzyme samples were recorded with almost identical spectra observed (Fig. 3), suggesting that modification does not result in gross changes of the enzyme conformation.
residues is the value of i which gives a straight line when plotting Ax/A0, the fraction of the fully active enzyme, against the number of isoindoles modified (m). The results in Fig. 4 show that the ALP activity is dependent upon formation of only one isoindole group.
Fig. 3 CD spectra of native and OPA-modified ALP from P. fucata. Far-UV spectra were recorded for native (ƹ) and OPA-modified (Ƹ ) enzyme at 25ć. Each spectrum represents the average of four independent scans.
Fig. 4 Correlation between the number of isoindole formed and the residual activity of ALP from P. fucata. Enzyme with a concentration 2.3 µmol/L was incubated with OPA at molar proportions from 1:20 to 1:1000. After incubation, the residual activity and the number of isoindoles formed were measured as described in the text. The data is presented as a Tsou’s plot for i = 1 (Ŷ), i = 2 (ż), and i = 3 (Ÿ).
2.2
Stoichiometry of OPA inactivation
The inactivated enzyme exhibited a new absorbance maximum at 337 nm, which is characteristic of isoindole absorption. An emission peak was also observed at 415 nm in the fluorescence emission spectrum excited at 337 nm. These results indicated that inactivation of the enzyme by OPA resulted in the formation of isoindole groups from the reaction of OPA and the enzyme[12]. The kinetic data showed that at most one isoindole derivative was produced per mole by the inactivation (1 mole isoindole was derived from 1 mole OPA). The limitations of kinetic methods in determining the number of modifiable sites essential for enzyme activity were described by Lery[11]. Therefore, the stoichiometry of the OPA inactivation was studied by quantitatively monitoring the isoindole formation with a Tsou’s plot analysis used to determine the number of isoindoles essential for the activity[13]. Assuming that the reactivities of all n reactive sites toward OPA are approximately equal and that modification of any one would inactivate the enzyme, the relationship between the residual activity, Ax, against residue modification will be ( Ax / A0 )1/ i
( n m) / n
(1)
where A0 is the initial activity. The number of essential
2.3
Double modification
The critical residues related to the formation of isoindole derivatives was investigated using double modification experiments. The enzyme was completely inactivated after incubation with 2.0 mmol/L DEPC (a regent for histidine modification) and lost 80% of the initial activity after pretreatment by 0.2 mmol/L TNBS (a regent for lysine modification). However, the enzyme retained 90% of its initial activity after incubation with 1.0 mmol/L DTNB for 2 h. Remodification of the DTNB (a regent for cysteine modification) modified enzyme by 1.0 mmol/L OPA caused complete inactivation. The native enzyme, enzymes modified by TNBS, DEPC, or DTNB did not present an emission peak at 415 nm in the fluorescence spectrum excited at 337 nm (data not shown), and therefore indicated no formation of isoindole derivatives. Figure 5 shows the effects of these residual specific modifiers on the isoindole fluorescence of the OPA modified enzyme. Complete premodification by DEPC or TNBS eliminated the characteristic isoindole derivative peak at 415 nm. However, pretreatment by DTNB did not prevent the formation of isoindole
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
418
fluorescence. These results strongly indicate that no cysteine residue was involved in the isoindole formation during the OPA inactivation. Thus, the inactivation was due to formation of an isoindole derivative involving lysine and histidine residues.
2.5
Affinity chromatography experiments
The substrate binding ability of the OPA-treated enzyme was evaluated on an ALP specific affinity column. Experiments were also conducted using the same amount of native enzyme. The elution graphs are shown in Fig. 6. The OPA-modified enzyme lost the ability to bind in the column and thus was directly washed out. However, the native enzyme could be captured by the column and then eluted with the competitive phosphate, which indicated the specific binding ability.
Fig. 5 Effect of lysine, histidine, and cysteine modifiers on the isoindole fluorescent of ALP from P. fucata. The enzyme (2.3 µmol/L) was incubated with 2.0 mmol/L DEPC, 0.2 mmol/L TNBS, or 1.0 mmol/L DTNB at 25°C for complete modification, respectively. The modified enzyme samples were then treated with 1.0 mmol/L OPA for 1 h. The change in the isoindole fluorescence was monitored at an excitation wavelength of 337 nm.
2.4
Protection experiments
The substrates 2-glycerol phosphate and adenosine monophosphate (AMP) were found to significantly protect the enzyme from inactivation by OPA. About 60% of the activity could be retained by incubating with 5 mmol/L 2-glycerol phosphate or 10 mmol/L AMP. Phosphate and tungstate, which are powerful competitive inhibitors, also markedly protected the enzyme against inactivation (Table 1). Table 1
Fig. 6 Affinity chromatography experiments for the modified ALP from P. fucata. The OPA-modified enzyme (a) and the native enzyme (b) were loaded onto an L-histidyldiazobenzylphosphonic acid agarose column which is specific to binding ALPs. An elution speed of 1 mLgmin–1 was applied and A280 was monitored for the eluted enzyme.
Inhibitors against OPA inactivation Residual Incubation system
activity (%)
Enzyme
100
+ OPA (200 µmol/L)
11
+ ȕ-GP (5 mmol/L) + OPA (200 µmol/L)
70
+ AMP (5 mmol/L) + OPA (200 µmol/L)
65
+ tungstate (10 mmol/L) + OPA (200 µmol/L)
61
+ phosphate (10 mmol/L) + OPA (200 µmol/L)
63
The values given are means of three independent sets of experiments with standard deviation less than 10%.
3
Discussion
OPA is a bifunctional cross-linking reagent that has been extensively used to detect the active site of a number of enzymes[14,15]. However, the modification of alkaline phosphatase, which is widely distributed in most living beings, by this reagent has not been widely investigated. The present studies showed that ALP from P. fucata could be irreversibly inactivated by OPA. The inactivation followed pseudo-first-order kinetics with the second-order inhibition rate constant
CHEN Hongtao ч܀ඁ et al˖Inhibition of Alkaline Phosphatase from Pearl Oyster ĂĂ
calculated to be 0.167 (mmol/L)–1gmin–1. The CD spectra of both native and modified enzymes were almost identical, indicating that the inhibition does not result in gross changes in the enzyme conformation and that inactivation was due to modification of essential residues at the active site. A combination of kinetic and statistical methods shows that the OPA inactivation is dependent upon formation of only one isoindole group. OPA is generally known to form an isoindole derivative by cross-linking thiol and amino groups[16]. However, DTNB titration of the OPA-modified enzymes showed no change in the number of thiol groups (data not shown). Furthermore, ALP from P. fucata was not inhibited by thiol reagents such as DTNB, indicating that thiol groups are not involved in the enzyme activity, which is consistent with ALPs from other sources[1,6,7]. Fluorescence studies indicated that modification of either lysine residue by TNBS or histidine residues by DEPC prevented formation of the isoindole derivative. Thus, these results prove the involvement of both lysine and histidine in the formation of the isoindole derivative. A proposed mechanism for the reaction of OPA with histidine and lysine residues[17] suggests that the aldehyde groups of OPA react with the secondary amine in the imidozole ring and the İ-amine in the lysine residue to form the fluorescent isoindole derivative. The importance of histidine residues in the catalytic activity of ALPs has been shown in many investigations. Histidines serve as ligands to the metals at the active site and are highly conserved in the active site of ALP from E. coli to humans[1,6,7]. Lysine residues are negatively charged and are potentially involved in anionic substrate binding[18,19]. In E. coli ALP, a lysine residue forms a secondary interaction to the substrate through a water molecule[20]. In Scylla serrata, a lysine residue is essential for the enzyme activity[21]. The inactivation of ALP from P. fucata by OPA strongly suggests the existence of histidine and lysine residues at or near the active site of this molluscan ALP. Protection experiments were performed to determine the effect of substrates and competitive inhibitors on OPA-induced inactivation. The results showed that 2-glycerol phosphate and AMP effectively protected the enzyme from inactivation, as well as the competitive inhibitors, phosphate, and tungstate. The
419
binding experiments confirmed that after the OPA modification, the enzyme lost the ability to bind with the specific affinity column. These results indicate that substrate binding can markedly affect the accessibility of OPA and the OPA modification can effectively prevent binding of the substrate, which leaves little doubt that the isoindole group formed by OPA inhibition is located at the substrate binding site of the ALP from P. fucata. The isoindole derivative formed in the enzyme active site could be used as an indicator to detect the polarity of the micro-environment of the active site[22]. There are many successful applications of OPA as a probe to ascertain the conformational flexibility of the active site[23,24]. Our studies demonstrated the formation of a fluorescent chemoaffinity label at the active site by OPA modification of ALP which can be used as a powerful tool to study the detailed structure-function relationship of the ALP of P. fucata to contribute to the study of the mechanism of marine biomineralization. Acknowledgements The author thanks Li Yu and Jia Wei for their enthusiastic help with the enzyme purification and Dr. Ma Zhuojun for his help with the oyster culture.
References [1] Kim E E, Wyckoff H W. Reaction mechanism of alkaline phosphatase based on crystal structures: Two-metal ions catalysis. J. Mol. Biol., 1991, 218: 449-464. [2] Stec B, Holtz K M, Kantrowitz E R. A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J. Mol. Biol., 2000, 299: 1303-1311. [3] Holtz K M, Kantrowitz E R. The mechanism of alkaline phosphatase reaction: Insights from NMR, crystallography and site-specific mutagenesis. FEBS Letter, 1999, 462: 7-11. [4] Mazorra M T, Bubio J A, Blasco J. Acid and alkaline phosphatase activities in the clam Scrobicularia plana: Kinetic characteristics and effect of heavy metals. Comp. Biochem. Phys. B, 2002, 131: 241-249. [5] Reddy M S, Rao K V. Methylparathion-induced alterations in the acethylcholinesterase and phosphatase in a penaeid prawn, Metapenaeus monoceros. Bull. Environ. Contam. Toxicol., 1990, 45: 350-357. [6] de Bacher M, McSweeney S, Rasmussen H B, Riise B W,
420
Tsinghua Science and Technology, August 2005, 10(4): 414̢420
Lindley P, Hough E. The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase. J. Mol. Biol., 2002, 318: 1265-1274.
[16] Quang K H. Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate
synthase
by
o-phthalaldehyde. J. Biol. Chem., 1990, 265: 6700-6704.
[7] le Du M H, Stigbrand T, Taussig M J, Menez A, Stura E A.
[17] George S P, Rao M B. Conformation and polarity of the
Crystal structure of alkaline phosphatase from human
active site of xylanase I from Thernomonospora sp. as
placenta at 1.8 Å resolution: Implication for a substrate
deduced by fluorescent chemoaffinity labeling. Eur. J.
specificity. J. Mol. Biol., 2001, 276: 9158-9165.
Biochem., 2001, 268: 2881-2888.
[8] Nilsen I W, Øverbø K, Olsen R L. Thermolabile alkaline
[18] Gite S, Reddy G, Shankar V. Active site characterization
phosphatase from Northern shrimp (Pandalus borealis):
of S1 nuclease: 1. Affinity purification and influence of
Protein and cDNA sequence analyses. Comp. Biochem.
amino-group modification. Biochem. J., 1992, 285:
Phys. B, 2001, 129: 853-861. [9] Xiao R, Xie L P, Lin J Y, Li C H, Chen Q X, Zhou H M,
489-494. [19] Richardson R M, Parés X, Cuchillo C M. Chemical
Zhang R Q. Purification and enzymatic characterization of
modification
alkaline phosphatase from Pinctada fucata. J. Mol. Catal.
cyclohexane-1,2-dione indicates the Lys-7 and Arg-10 are
B Enzym., 2002, 17: 65-74.
involved in the P2 phosphate-binding subsite of bovine
[10] Simons S S Jr, Johnson D F. Reaction of o-phthalaldehyde and thiol with primary amines: Formation of 1-alkyl (and aryl) thiol-2-alkylisoindoles. J. Org. Chem., 1978, 43: 7769-7775. [11] Lery H M, Leber P A, Ryan I M. Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphate and other phosphate compounds. J. Biol. Chem., 1963, 238: 3654-3659. [12] Harris G W, Jenkins J A, Connerton I, Pickersgill R W. Defined crystal structure of the catalytic domain of xylanase A from Pseudomonos fluorecens at 1.8 Å resolution. Acta Cryst., 1996, D52: 393-401.
by
pyridoxal
5’-phosphate
and
pancreatic ribonuclease A. Biochem. J., 1990, 267: 593-599. [20] Sun L, Carolan D C, Kantrowitz E R. Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center. Biochemistry, 1999, 38: 2842-2848. [21] Chen Q X, Zhou H M. An essential lysine residue of green crab (Scylla serrata) alkaline phophatase. Biochem. Mol. Bio. Int., 1998, 46: 225-231. [22] Palczewski
K,
Hargrave
P
A,
Kochman
M.
o-Phthalaldehyde, a fluorescence probe of aldolase active site. Eur. J. Biochem., 1983, 137: 429-435.
[13] Tsou C L. Relation between modification of functional
[23] Wang Z F, Xu Y K, Zhou H M. Kinetics of irreversible
groups of proteins and their biological activity: 1. A
inhibition of creatine kinase during modification by
graphical method for the determination of the number and
o-phthaldehyde. Enzyme Protein, 1995, 48: 1-9.
type of essential groups. Sci. Sin., 1962, 11: 1535-1558.
[24] Heng Y B, Wang Z, Chen B Y, Wang X C. Multiple
[14] Anjali P, Saifuddin S, Sarvagya K S. Identification of
effects of chemical reagent on enzyme: o-Phthalaldehyde-
cysteine and lysine residues present at the active site of
induced inactivation, dissociation and partial unfolding of
beef liver glutamate dehydrogenase by o-phthalaldehyde.
lactate dehydrogenase from pig heart. Int. J. Bio.
Biochim. Biophys. Acta B: Pro. Struc. Mol. Enzymol., 1996,
Macromol., 2003, 32: 191-197.
1293: 122-128. [15] Ahn J Y, Choi S, Cho S W. Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde. Biochimie, 1999, 81: 1123-1129.