Phytochemtstry,Vol. 31, No. 5, pp. 1529-1532,1992
0031-9422/92%5.00+0.00 0 1992Pergamon Press plc
Printed in Great Britain.
OF MAIZE PHOSPHOENOLPYRUVATE BY TETRANITROMETHANE
GURURAJ B. MARALIHALLI and ANIL S. BHAGWAT* Molecular Biology and Agricultural Division, Bhabha Atomic Research Centre, Trombay, Bombay-400085, India (Receioed 30 July 1991) Key Word Index-Zea
mays; Gramineae; maize; PEP carboxylase; tyrosine residues; tetranitromethane.
Abstract-Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of tetranitromethane. The inactivation did not follow any of the known kinetic mechanisms. The inactivation resulted from the specific modification of one tyrosine residue per enzyme protomer, although sulphydryl groups were also modified by the reagent. This conclusion is based on the pH dependence of the inactivation and also on studies done after protecting the free -SH groups of the enzyme with p-hydroxymercuribenzoate. The substrate PEP and Mg2+ offered almost complete protection against tetranitromethane inactivation. Many other effecters of the enzyme also gave substantial protection, indicating the modification at or near the active site.
The use of reagents for specific modification of amino acid residues in proteins has been a fruitful means of determining the chemical structure of the ligand binding sites. This approach is particularly interesting with the regulatory enzymes, where both the allosteric interactions and the catalytic process can be selectively affected. Phosphoenolpyruvate carboxylase [orthophosphate: oxaloacetate carboxylase (carboxylating), EC 184.108.40.2061 catalyses irreversible carboxylation of PEP to yield oxaloacetate and inorganic phosphate. The reaction is highly exergonic. The enzyme, which was discovered in plant systems by Bandursky and Greiner , is present in all plants and is apparently absent in animal systems. The maize PEP carboxylase has an M, of 400000 and is a homotetramer. Topography of the active site of maize PEP carboxylase has been studied using various compounds having structural similarities with PEP [2-51. The chemistry of the active site of PEP carboxylase has been elucidated by chemical modification studies using phenylglyoxal , eosine isothiocyanate , pyridoxal phosphate , o-phthalaldehyde  and diethylpyrocarbonate [lo]. It has been suggested that tyrosyl side chains of enzymes may be involved in either stabilizing the structure of proteins by means of internal hydrogen bonding with side chains such as aspartic or glutamic acid, or is affecting the activity by ionization of the phenolic hydroxyl group [l l-131. Several methods for determination of the degree of tyrosyl residue exposure to the solvent have heen employed [14,15-J. Products from the reaction
*Present address: Institut fiir Biocbemie und Molekulare Biologic, Techniscbe Universitiit Berlin, Franklinatrah-29,1000 Berlin-lo, Germany.
with tetranitromethane have been used to locate this residue and determine its importance in enzyme activity
Chemical modification was chosen as a probe to identify essential amino acid residues at the active site of this enzyme. In this study, we have tried to establish that tyrosine residues are essential for activity of maize PEP carboxylase. The loss of activity cannot be correlated with the oxidation of -SH groups, as they were masked. RESULTS AND DISCUSSION
Inactivation of PEP carboxylase by treatment with TNM PEP carboxylase was inactivated to almost 80% by incubation with an IO-fold molar excess of TNM for 2 min at 30” (Fig. 1). At ca 150-fold molar excess of TNM, the inactivation of the enzyme was almost complete. The kinetics of inactivation of the enzyme by TNM are, however, very complex. A similar observation has been reported with many other enzymes, where TNM was used to demonstrate the involvement of tyrosine residues in catalysis. Our initial experiments showed that the -SH groups of the enzyme were also oxidised simultaneously with the nitration of tyrosine residues. As -SH groups are essential for PEP carboxylase activity [ 171, it became necessary in all the experiments reported in this paper to reversibly protect the free ‘-SH groups of the enzyme with phydroxymercuribenzoate before TNM modification. Amino acid residue responsible for TNM inactivation In order to clarify the above point further, the following experiments were carried out. Nitration of tyrosine is dependent on pH, with little or no modification occurring at ca pH 6. The nitration and thiol oxidation can thus be
by performing the reaction at pH 6. The
G. B. MARALIHALLI and A. S. BHAGWAT
Fig. 1. Time course of inactivation of the enzyme at different TNM concentrations. The enzyme (2.74 /tM in 0.05 M Tris-HCl, pH 7.9) was incubated with the indicated concentrations of TNM at 30”. At indicated time intervals a small aliquot was quenched with 10 mM dithiothreitol and assayed for the enzyme activity. The control received an equal amount of ethanol. (0), 50/.~M; (0). 75 PM; (a), 1OOpM; (A), 150j~M; (Z), 2OOpM TNM.
Fig. 2. Effect of pH on TNM modification of the enzyme. The enzyme, in either 50 mM HEPES-NaOH (pH 6.3) or Tris-HCI (pH 7.9), was treated with the indicated concentrations of TNM for 8 min. Controls without TNM had equal amounts ofethanol. (0). pH 6.3; (O), pH 7.9.
Table 1. Effect of sulphydryl groups on TNM inactivation Activity pHMB treatment
after before D’IT treatment
The masking of thiol groups was performed by incubation of the enzyme with 100 PM p-hydroxymercuribeznzoate for 15 min at 2.5” in 25 mM HEPESNaOH (pH 7). Tetranitromethane treatment was carried out by incubation of the enzyme (1.6 FM) with 50-fold molar excess of TNM for 12 min at 30”. The enzyme activity was measured before and after treatment with 25 mM dithiothreitol at room temperature for 15 min and 2 hr.
extent of inactivation by TNM increased as the pH increased from 6.3 to 7.9 (Fig. 2). The slow inactivation at pH 6.3 possibly eliminates the participation of thiol oxidation in TNM inactivation. To substantiate this point further, the -SH groups of the enzyme were masked with p-hydroxymercuribenzoate before TNM modification. If the inactivation was due to thiol oxidation rather than modification of tyrosyl residue, full enzyme activity should appear after regeneration of -SH groups by treatment with dithiothreitol. If thiol oxidation was not responsible for TNM inactivation, the enzyme once treated with TNM would not recover its activity upon treatment with dithiothreitol. Very little enzymic activity reappeared after incubation with 25 mM dithothreitol
(Table 1). In contrast, the control without TNM showed ca 50% recovery of the enzyme activity. Even though one exposed and two buried (detected after SDS treatment) -SH groups were modified by TNM (data not presented), it seems that inactivation of the enzyme was not due to -SH modification. Spectral characteristics of TNM modijied enzyme
Modification of the enzyme with TNM at pH 7 caused spectral changes which are consistent with the nitration of tyrosine residues . A difference spectrum of the modified versus control enzyme showed a broad peak centred around 430 nm.
Modification of maize PEP carboxylase Protection of PEP carboxylase against TNM inactivation
The results presented so far have indicated that the loss of activity was due to the modification of tyrosyl residues of the enzyme. In order to find out whether the essential tyrosine residues are located at or near the active site of the enzyme, the protective effects of substrate and other effecters on the rate of inactivation by TNM were studied. The reactive tyrosines are indeed located at or near the active site of the PEP carboxylase (Table 2). PEP + Mg2 + offered substantial protection while PEP alone at 30 mM offered 50% protection. The other effecters of the enzyme were less beneficial. The protective effect of PEP + MgZ + was clearly concentration dependent. A LineweaverBurk plot gave an apparently straight line with a k1,2 protection value of 2.4 mM for PEP + Mg2 ‘. Such plots have been used earlier to show protection by substrate and effecters against photoinactivation by PLP and TNM modification of tryptophanase [19, 203.
lar size or subunit structure such as interlinking of the subunits. Higher levels of TNM have been previously shown to cross link subunits of some multimeric enzymes . In the present studies, 60-80-fold molar excesses of TNM were used, which is an order of magnitude lower than the concentrations used for interlinking of subunits. From the stoichiometry data it is suggested that only one tyrosine plays a role in catalysis by maize PEP carboxylase. The actual functional role of this residue cannot, however, be elucidated from the present data. It seems likely that nitrotyrosine causes delocalization of the charges at or near the active site, either by enhancing the ionization of the phenolic proton or by increasing the charge density due to high electronegativity of the attached group. If this is true, then the phenolic proton could be involved in a critical hydrogen bond or could act as a proton donor during catalysis. EXPERIMENTAL
Stoichiometry of TNM modification
Measurements of A at 428 nm along with the determination of the residual enzyme activity show that PEP carboxylase incorporated about 0.65 mol nitrotyrosine per mol of enzyme protomer when extrapolated to 100% inactivation. The enzyme protected with PEP + Mgz+ showed correspondingly reduced nitrotyrosine formation (data not included. With respect to the molecular properties of the enzyme modified with TNM there are unwanted side reactions which may alter the properties of the enzyme, e.g. formation of dinitrotyrosine  or reaction with nitrous acid (a decomposition product of TNM) . Each of them may cause polymerization of the protein. To check the possibility that the inactivation by TNM was not due to polymerization or destruction of the subunit structure, sodium dodecylsulphate polyacrylamide gel electrophoresis on TNM modified enzyme and the untreated control was done. The samples were reduced with 2-mercaptoethanol before they were subjected to the electrophoresis. The TNM modified enzyme showed an identical mobility to that of the untreated control (data not presented). From this it is evident that the main process of TNM inactivation does not involve any changes in the molecu-
Table 2. Protection of maize PEP carboxylase against TNM inactivation Additions None Phosphoenolpyruvate PEP+Mg*+ Mg’+ Glucose-6-phosphate p_Glycolate Malate Glycine
% of control
30 30+10 10 10 10 10 10
15 49 9 23 38 30 31 21
The percentage activity remaining was compared with a control lacking TNM but containing the same amount of eflector and ethanol. The enzyme (2.8 PM) was modified with 50fold molar excess of TNM for 5 min after preincubation with each effector for 5 min.
Pur$cation of the enzyme. PEP carboxylase was purified to homogeneity from maize leaves essentially by the method of ref.  with slight modification in the extraction buffer and fractionation. Protein determination and enzyme assay. Protein concn was estimated by measuring A at 280 nm assuming A 280 nm of 1=0.88 mg ml-’ and also by the method of ref.  after removal of the reducing agent by Sephadex G-25 column. The enzyme activity was measured by a linked assay using malate dehydrogenase. The reaction mixture (I ml) contained HEPES,
100mM (pH 80); MgCI,, 2 mM; MDH, 4 units; PEP, 2 mM; NADH, 1OOyM; and the enzyme. The enzyme activity was monitored by change in A at 340 nm at room temp. (25“).The sp. act. of the enzyme was 14-16 pmol of oxaloacetate formed per mm per mg protein. Dilutions of TNM stock soln were made in EtOH and commercial TNM was taken as 8.4 M. Nitrotyrosine was deteo ted at 428 nm using an extinction coefficient of 4100 M-’ cm-’ at pH 9.5. Modification of PEP carboxylase by TNM. The enzyme (2.5-4 PM in 50 mM Tris-HCl, pH 7.9) was treated with 80-lOOfold molar excess of TNM in 95% EtOH to yield a final EtOH concn of l-2%. This mixture was kept at 30” for various periods. The reaction was terminated by addition of 10 mM dithiothreito1 (final concn). A small aliquot was used for the activity measurement. Sulphydryl group analysis. The method of ref.  was used to determine the exposed and total sulphydryls. The enzyme was completely denatured with 5% SDS to determine total sulphydry1 groups. Chemicals. PEP (trisodium salt), DTNB, tris, dithiotreitol and HEPES were obtained from Sigma. Tetranitromethane was a product of Spectrochem (India). Acknowledgement-The authors thank Prof. Joachim Vater (Institiit fiir Biochemie und Molekulare Biologic, Technische Universitiit Berlin) for critically reading the manuscript.
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