May 27, 1981 - James Om$ and Robert Haselborn§. From the Department of Biophysics and Theoretical Biology, The Unioersity of Chicago, Chicago, Illinois ...
THEJOURNALOF BIOLOGICAL CHEMISTRY
No.24, Issue of December 25, pp. 13099-13104.1981 Printed m U.S.A Vol. 256,
Kinetic and Inhibition Studiesof Glutamine Synthetase from the Cyanobacterium Anabaena 7120* (Received for publication, May 27, 1981)
James Om$ and Robert Haselborn§ From the Department of Biophysics and Theoretical Biology, The Unioersity of Chicago, Chicago, Illinois 60637
A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined.Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 m,respectively; that for ammonia was found to be below 20 PM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.
The enzyme glutamine synthetase (L-g1utamate:ammonia ligase (ADP-forming), EC 6.3.1.2) plays a crucial role in the nitrogen metabolism of all prokaryotes, being responsible for the assimilation of ammonia' and for the formationof glutamine, a compound that stands as thebeginning of numerous biosynthetic pathways (1).The kinetic properties of the enextensively (2). zyme from Escherichia coli have been studied In that system, the activity of the enzyme can be regulated in two ways. One involves covalent modification: the additionof an AMP moiety to each subunit (3, 4). Fully adenylylated glutamine synthetase is inactive in the biosynthetic reaction:
moniaassimilation (8). Itis similar in molecularweight, quaternary structure, and amino acid composition to the E . coli glutamine synthetase; its NHz-terminal amino acid sequence is homologous to that of the E. coli enzyme (7). However, the Anabaena enzyme is not subject to adenylylation. This statement is based on direct observation in Anabaena: cells labeled with 'lP under a variety of conditions E. coli yielded unlabeled which produce 32P-labeled enzyme in 'enzyme in Anabaena? Second, when theAnabaena gene for glutamine synthetase was cloned and transferred to E. coli, again the gene product was found to be unlabeled under conditions that produced highly labeled E. coli enzyme (9). Anabaena must regulate the activityof glutamine synthetase under one known circumstance: the symbiotic association with the fern Azolla (10). There, the Anabaena glutamine synthetase activityis reduced by a mechanism which permits the ammonia producedby nitrogen fixation in the Anabaena cells to be exported for the use of the hostfern. In view of the structural similarity tothe E . coli enzyme, the needfor regulation, and the absenceof adenylylation, we undertook a study of the kinetic parameters of the Anabaena enzyme and the effects on them of a variety of possible effectors. MATERIALS AND METHODS
Anabaena 7120 was grown, glutaminesynthetase was purified, and activities were assayed as described previously(7),including the final purification step of affinity chromatography on blue Sepharose. The biosynthetic glutaminesynthetaseassay used wasthat of Shapiro and Stadtman ( l l ) run , at 22 "C, in which ATP isconstantly regenerated, and its consumption is monitored, with a pyruvate kinase/Iactate glutamine + ADP + P, (1) dehydrogenase couple. Absorbance at 340 nm was continuously reGlutamate + NH,' + ATP i corded and activities were estimatedfrom the curves afterlag phase. Mg2+ Substrate concentrations in the standard assay were glutamate, 36 but retains activity in a nonphysiological reaction (transferase mM; ATP, 3 mM; and ammonia, 50 mM. The pH dependence of the biosynthetic reaction was determined activity) (5): over the range pH6.5-9.0. A broad optimumwas observed, decreasing Glutamine + NH, OH y-glutamyl hydroxamate (2) to 40% activity at pH 6.5 and 50% at pH 9. All of the experiments ~ ~ 0 i 3 - ADP, , M~J+ with the biosynthetic reaction reported here were done at pH 7.60 and 22 "C. Linearity of the biosynthetic assay with respect to enzyme Glutamine synthetases from E . coli and other batteria are concentration was observed beyond 25 nmol/min of ATP consumed. also subject toallosteric inhibition by a wide array of effectors All of the inhibition studieswere made within this range. that correspond to the end products of biosynthetic pathways: The transferase assay used was that of the same reference (11), with Mn2+ loweredby a factor of 10 topreventprecipitation of amino acids, nucleotides, and other compounds (2,6). We have recently purified glutamine synthetase from t,he Mn3(As0&.It was performed at 37 "C. Protein was estimated according to Bradford (12), with the assay filamentous, nitrogen-fixing cyanobacterium Anabaena 7120 by acid hydrolysis and amino acidanalysis of an aliquot ( 7 ) . In this organism, the enzyme is responsible for all am- standardized of the protein.
* This research was supported by Grant GM 21823 from the United States Public Health Service and Grant 5901-0410 from the United States Dept of Agriculture/Science and Education Administration through the Competitive Grants Office. +Supported by United States PublicHealthServiceTraining Grant GM-780. § To whom correspondence should besent. In this paper, the term "ammonia" refers to free ammonia plus ammonium ion, the latter being the dominant form throughout the pH region involved.
RESULTS
Turnover Number-Purified Anabaena glutamine synthetase (1.76 pg) was found to catalyze the consumption of 28.9 m o l of ATP/min in the biosynthetic assay,indicatinga turnover number of 10,000 per 12-subunit molecule ( M , = 610,000). For the transferase reaction, with Mn2+ cofactor, as
13099
J. Orr and R. Haselkorn, unpublished results.
Anabaena Glutamine Synthetase: Kinetics and Inhibition
13100
y-glutamyl hydroxamate was produced at a rate of 214 pmol/ mg of enzyme/&, corresponding to a turnover number of 131,000. These values are compared with those for other prokaryotic glutamine synthetases in Table I. The turnover numbers in the biosynthetic reaction are similar for all of the enzymes except that from E. coli, although the temperature at which the Anabaenavalue is determined is lower than that for the other species. The transferase values are also similar, those cases where the values are lower may be due to failure to adjust theMn" concentration appropriately. Michaelis Constants-Apparent Michaelis constants were the biosynthetic determined for each of the three substrates in reaction. In the cases of glutamate (Fig. 3, below) and ATP (Fig. l),double reciprocal plots were linear, allowing unambiguous determination of K, for each. These were 2.1 and 0.32 m, respectively (Table I). For subsequent inhibition studies, standard concentrations used for glutamate and ATPwere 36 and 3 m, respectively, The K,,,for ammonia appeared very low, and so could not be measured in the same way as the K, values for the other substrates. It was, however, possible to put an upper bound on it, as follows. A small amount of ammonia was added to an assay mixture lacking only ammonia. The absorbance was monitored as thereaction proceeded to equilibrium (Fig. 2). The substrate saturated rate (V,& was seen as the initial rate. We intended to take the ammonia concentration as K, at that point where the instantaneous velocitywas equal to ImV,,. The ammonia concentration at that point was to be estimated by comparing the absorbance with the absorbance at equilibrium. The differencewould then represent the amount of NADH consumed before equilibrium was reached; this wouldbe equal on a molar basis to the amount of ammonia consumed. Taking the ammonia concentration at equilibrium as zero gives us the ammonia concentration at any nonequilibrium point directly. Various technical difficulties, discussed below, prevented
our using this approach to obtain an exact value for K , for ammonia. We instead were able to place successive upper limits on this parameter. To begin, in Fig. 2, we note that the first point where the glutamine synthetase curve deviates noticeably from a straight line (point a, at56 pM) is clearly an upper bound for K,. Until the ammonia concentration drops to this point, it is sufficient to saturate theenzyme. This approach rests on four assumptions: first, that the ammonia concentration is zero at equilibrium; second, that the reaction rate is always what Michaelis-Menten theory
I ATP J "
FIG. 1. Lineweaver-Burk plot of glutamine synthetase reaction velocity as a function of ATP concentration. Other reagents were as in the standard assay. The straight line (x-intercept = -1/0.32 mM") is a hyperbolic best fit to the data. Three mM ATP (0)was used for the standardassay. \\
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TABLE I Parameters of glutamine synthetase from various prokaryotic sources Michaelis constanta Species
NR'
Glutamine
Specific activities' Biosynthetic
'hnsferase
10,020
131,000
Reference
mM
Anabaena
