Plant Cell Reports (1985) 4:206-209
Plant Cell Reports © Springer-Verlag 1985
Evidence for the importance of histidine at the active site of argininosuccinate synthetase from soybean Peter D. Shargool Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Received May 1, 1985 / Revised version received June 12, 1985 - Communicated by F. Constabel
MATERIALS AND METHODS
Studies to determine the role of histidine in catalysis by L-argininosuccinate synthetase (EC 184.108.40.206) were carried out with the enzyme isolated from soybean cell suspension cultures. These experiments utilized analogues of the substrates citrulline and aspartate to investigate substrate binding, and to determine which portion of the molecule were required for binding at the active site of the enzyme. Photooxidation studies using rose bengal were carried out to define the importance of histidine residues for catalysis. These studies suggest that an active site histidine residue has an important role to play in the formation of argininosuccinate by this enzyme.
The enzyme argininosuccinate synthetase has an important role to play in the synthesis of arginine in plants as well as other organisms (Shargool and Cossins 1969, Shargool 1973). The most recent studies by Raushel and Seiglie (1983) on argininosuccinate synthetase isolated from bovine liver, have looked at the kinetic mechanism of the enzyme. The results of this study confirmed the earlier suggestion made by Rochovansky and Ratner (1967) that the function of the ATP used in the reaction is to activate citrulline to form an enzyme bound adenylylcitrulline intermediate, which is subsequently attacked by aspartate to form argininosuccinate (Raushel and Seiglie 1983). No studies have yet been published that have looked at the way in which the active site of the enzyme is involved in the formation of adenylylcitrulline. This communication describes experiments using homogeneous preparations of enzyme obtained from plant cell cultures, and various analogues of citrulline and aspartate, to determine the necessity of various parts of these substrate molecules for binding and catalysis. Together with the results of rose bengal catalyzed photooxidation experiments, this work has led to the suggestion that one or more histidine groups play an important role in catalysis by argininosuccinate synthetase.
All compounds used as analogues of citrulline or aspartate, were obtained from Sigma Chemicals Co., or Calbiochem. Rose bengal was obtained from Serva Fein Biochemica Ltd., and was further purified by the method of Brand et al (1967). The processed rose bengal yielded a single spot on thin layer chromatography, using the methods of Kaye and Weitzman (1967). Plant Cell Cultures,
Suspension cultures of Soybean (Glycine Max L. var. Mandarin) were grown, and processed to yield argininosuccinate synthetase, using previously described methods (Shargool 1971, 1973). This yielded a homogeneous enzyme preparation that gave one band on electrophoresis in polyacrylamide gel, and staining with Coomassie blue dye (Shargool 1971). T ~ argininosuccinate synthetase assay using carbamyl-- C-citrulline has been described previously (Shargool 1969, 1973). Rose Bengal
Photooxidation was carried out using a procedure esentially similar to that described by Ray and Koshland (1962). Samples for irradiation contained 6-15 mg of protein, and 0.01% (w/v) rose bengal in 3.3 ml volumes of enzyme assay buffer (0. i M pH 7.8 Tricine). Illumination was provided by a single 150 watt spot light, placed at a distance of i0 cm below the sample. The temperature of the water surrounding the sample was kept at i0°C throughout the photooxidation. Aliquots of 0.2 ml volume were taken for assay; each assay was carried out in tubes that were foil wrapped to exclude light. Kinetic data Kinetic data on the type of reaction catalyzed, also Km and Ki values, were obtained by keeping the concentration of one substrate constant, while the concentrations of the other two were varied as described by Cleland (1970). The kinetics presented are based on the mean values of at least three independent experiments. Lines were fitted to data points of graphs using linear regression programs.
207 RESULTS AND DISCUSSION
Mechanism and Michaelis constants All double reciprocal plots obtained, were linear and intersected to the left of the ordinate indicating a sequential reaction mechanism. Km values obtained for each of the three substrates were aspartate
1.2xlO-4M, citrulline 2.6xlO-4M, and
ATP 1.0xl0-4M. All of these findings are very similar to results obtained using enzyme prepared from bovine liver (Rochovansky et al., 1977, Raushel and Seiglie 1983), and suggest a close similarity between argininosuccinate synthetase enzymes from plant and animal sources. Inhibition experiments The list of analogues of citrulline and aspartate utilized in this study is shown in Tables 1 and 2, together with a description of the structural difference between each analogue and either citrulline or aspartate. The results of the inhibitor studies carried out with analogues of citrulline (Table i) indicate that modifications of the citrulline molecule involving the carbamyl group, give compounds that are still competitive with citrulline, each with similar Ki's° Thus it would appear that the carbamyl group of citrulline is not involved in binding of citrulline to the active site of the enzyme, although this group is involved (together with the amino group of aspartate) in the formation of argininosuccinate.
The conditions governing the binding of aspartate (Table 2) appear even more stringent than those governing that of citrulline. All parts of the aspartate molecule appear essential for binding, since Ki values could not be recorded for most of the analogues utilized. A very high Ki value was recorded for L-Aspartyl $-hydrazide, indicating that this modification interferes less with binding than does ~-methyl ester formation. Photooxidation studies Experiments utilizing rose bengal sensitized photooxidation of histidine residues in argininosuccinate synthetase, were carried out in the presence and absence of substrate levels of aspartate or aspartate plus ATP. The results of such experiments are shown in Fig. 1. It can be readily seen that photooxidation in the absence of substrates gives a curve with complex dimensions when plotted on semilog paper. In contrast, photooxidation in the presence of aspartate and ATP, or aspartate alone, yields a simple straight line in the same type of plot. The amount of enzyme activity lost during photooxidation for 5 minutes, is 35% in the presence of aspartate and ATP, or aspartate alone, compared with 68% in the absence of these substrates. The results described in this communication suggest that histidine residues in argininosuccinate synthetase, may have importance in binding and catalysis. Photooxidation of the enzyme in the absence of substrates can be seen from Fig. 1 to yield a complex curve. This type of data is similar
INHIBITORY PROPERTIES OF VARIOUS CITRULLINE ANALOGUES
Portion of Citrulline Molecule Altered
Type of InhibiOion Displayed
5 x 10 -4
3.5 x 10 -4
5 x 10 -4
backbone methylene group substituted
N-Acetylarginine carbamyl and R-amino groups
carbamyl and ~-carboxyl groups
L-Arginine ethyl ester
carbamyl and ~-carboxyl groups
When portions of the citrulline molecule other than the carbamyl group are modified, then the ability to bind to argininosuccinate synthetase is eliminated in most cases. The weak binding seen in the case of arginine ethyl ester, presumably indicates that this compound still resembles citrulline sufficiently for it to bind weakly. When the carboxyl group is completely removed from arginine to yield agmatine, then the compound is not able to act as an inhibitor, indicating the carboxyl group is indeed important for binding to take place.
9 x 10 -4
to that obtained by other workers, such as Ray and Koshland (1962), for photooxidation of histidine residues in enzymes. These workers after extensive mathematical analysis of their data suggest that the simplest explanation of such curves is the existence of two types of histidine residues, easily accessible, and inaccessible. In the data of fig. I, it is thought that the photooxidation reaction -I with a rate constant of k = 0.25 min may be for accesible histidine residues, the reaction with a
k = O.(~,6min -1
t l •
) 9 m i n -1
,I I | I
I I t
I I I I I I I I
I I I t I I
Time ( m i n i
Fig. I. Rose Bengal sensitized photooxidation of enzyme. Enzyme plus rose bengal ~ ~ , enzyme plus rose bengal and either aspartate alone, or aspartate plus ATP = = , enzyme plus either aspartate, or aspartate and ATP ~ . Samples for each experiment contained 6-15 mg of protein, and either 0.01% rose bengal alone, or substrates alone, or combinations of rose bengal plus aspartate and ATP. The aspartate and ATP were each present in 3-5 mM concentrations.
209 TABLE 2.
THE INHIBITORY PROPERTIES
Portion of Aspartate Molecule Altered
Type of Inhibition Displayed
Competitive (Ki = 7.1 x 10-3M)
Extra methylene group in backbone
rate constant of k = 0.12 min -I, for less accessible residues. The rate constant for photooxidation in the presence of substrates has a value of k = 0.09 •
OF VARIOUS ASPARTATE ANALOGUES
mln , which is close to the rate constant seen for less accessible residues. Thus it is proposed that the more accessible histidine residue (or residues) of argininosuccinate synthetase, may be actively involved in enzyme catalysis. This type of evidence relies heavily on histidine residues in argininosuccinate being modified in preference to other residues. Various authors have indicated in the past that rose bengal is a photooxidizing reagent with specificity for histidine residues (Bellin and Yankus, 1968; Westhead, 1965; Martinez-Carrion, 1967). In spite of this work, it should be noted that various amino acid residues other than histidine can be modified by photooxidation. Thus in order for this study to be rendered definitive, peptide mapping and amino acid analysis to demonstrate exactly which residues are modified should be carried out. It is also noteworthy that support for the involvement of an uncharged, imidazole ring in catalysis by argininosuccinate synthetase is seen in the pH activity curve published by Shargool and Cossins (1969). Maximal activity was seen between pH 7.4 and 8.0, and the mid point of the acid limb of the curve fell at 6.4. This would be consistent with the involvement of an uncharged histidine residue in the catalytic process. If the two sets of data presented in this communication are considered jointly, then a number of mechanistic schemes can be devised for the involvement of a histidine residue in either the formation of an adenylylcitrulline intermediate, or in argininosuccinate formation. It is felt that additional data analysing the active site of the enzyme should be obtained before such mechanisms appear authentic.
ACKNOWLEDGEMENTS The author would like to acknowledge of research from NSERC of Canada.
a grant in aid
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