scholarly journals Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5′-phosphate

1978 ◽  
Vol 173 (1) ◽  
pp. 53-58 ◽  
Author(s):  
I A Hemmilä ◽  
P I Mäntsälä
Keyword(s):  
Enzyme Activity ◽  
Glutamate Dehydrogenase ◽  
Bacillus Megaterium ◽  
Glutamate Synthase ◽  
Complete Loss ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Complete Protection ◽  
Complete Inactivation ◽  
Arginine Residues

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5′-phosphate and this inactivation increased 3–4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5′-phosphate.

scholarly journals Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae

1991 ◽  
Vol 279 (1) ◽  
pp. 135-139 ◽  
Author(s):  
A K Robinson ◽  
L D Barnes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Modification ◽  
Binding Site ◽  
High Specificity ◽  
Order Rate Constant ◽  
Complete Loss ◽  
Arginine Residue ◽  
First Order ◽  
Arginine Residues ◽  
Pseudo First Order

Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner. The second-order rate constant was 11.5 +/- 2.5 M-1 min-1. The loss of activity was a linear function of the incorporation of [7-14C]phenylglyoxal. The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity. The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi. The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not. Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively. The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal. Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A.

scholarly journals Modification of an arginine residue in pig kidney general acyl-coenzyme A dehydrogenase by cyclohexane-1,2-dione

1982 ◽  
Vol 207 (3) ◽  
pp. 415-419 ◽  
Author(s):  
Z Y Jiang ◽  
C Thorpe
Keyword(s):  
Residual Activity ◽  
Competitive Inhibitor ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Excess Substrate ◽  
Arginine Residues ◽  
Modified Enzyme ◽  
High Concentrations ◽  
Acyl Coenzyme A

The flavoenzyme pig kidney general acyl-CoA dehydrogenase (EC 1.3.99.3) is inactivated by cyclohexane-1,2-dione in borate buffer in a reaction that exhibits pseudo-first-order kinetics. Strong protection is afforded by the substrate octanoyl-CoA, as well as by heptadecyl-CoA, a potent competitive inhibitor of the dehydrogenase that does not reduce enzyme flavin. Enzyme exhibiting 10% residual activity in borate buffer contains about 1.3 modified arginine residues per flavin molecule. Very little reduction of the modified enzyme in borate buffer occurs at high concentrations of octanoyl-CoA, in marked contrast with the stoicheiometric reduction of the native enzyme. However, in phosphate buffer alone, the modified enzyme exhibits 55% residual activity and, although binding of substrate is still seriously impaired (apparent Kd=14 microM), excess substrate effects the formation of the characteristic reduced flavin X enoyl-CoA charge-transfer complex. These results suggest that the susceptible arginine residue, though not catalytically essential, is probably within the acyl-CoA-binding site of general acyl-CoA dehydrogenase.

scholarly journals Evidence for the presence of an essential arginine residue in photoreactivating enzyme from Streptomyces griseus

1985 ◽  
Vol 229 (2) ◽  
pp. 469-476 ◽  
Author(s):  
A P M Eker
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Streptomyces Griseus ◽  
Enzymic Activity ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Dna Binding Site ◽  
Arginine Residues ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Butane-2,3-dione inhibits the enzymic activity of Streptomyces griseus photoreactivating enzyme (PRE). Some characteristics of the inhibition, notably the enhancement by borate buffer and the reversibility, indicate that arginine residues are modified. From the kinetics of inhibition it can be concluded that a single essential arginine residue is involved. U.v.-irradiated DNA, the substrate for PRE, protects the enzyme against inactivation by butane-2,3-dione. This suggests that the essential arginine residue is situated in or near the u.v.-irradiated-DNA-binding site. Non-irradiated DNA at higher concentrations also protects against inactivation, indicating that PRE can form non-specific complexes. From the ratio of complex constants obtained from protection experiments with non-irradiated and u.v.-irradiated DNA it appears that PRE preferably binds to dimer sites.

scholarly journals Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium

1978 ◽  
Vol 173 (1) ◽  
pp. 45-52 ◽  
Author(s):  
I A Hemmilä ◽  
P I Mäntsälä
Keyword(s):  
Escherichia Coli ◽  
Glutamate Dehydrogenase ◽  
Bacillus Megaterium ◽  
Main Product ◽  
Glutamate Synthase ◽  
Cross Linking ◽  
Purification And Properties ◽  
Glutamate Synthetase ◽  
Dependent Activity ◽  
Dimethyl Suberimidate

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.

scholarly journals Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver

1981 ◽  
Vol 195 (3) ◽  
pp. 735-743 ◽  
Author(s):  
S Ramakrishna ◽  
W B Benjamin
Keyword(s):  
Rat Liver ◽  
Active Site ◽  
Arginine Residue ◽  
Atp Citrate Lyase ◽  
First Order ◽  
Complete Inactivation ◽  
First Order Kinetics ◽  
Citrate Lyase ◽  
Arginine Residues ◽  
Pseudo First Order

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

Ammonia, glutamine, and asparagine: a carbon–nitrogen interface

1988 ◽  
Vol 66 (10) ◽  
pp. 2103-2109 ◽  
Author(s):  
K. W. Joy
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Higher Plants ◽  
Amino Nitrogen ◽  
Glutamate Synthase ◽  
Dinitrogen Fixation ◽  
Similar Function ◽  
Metabolic Processes ◽  
Organic Form ◽  
Primary Input

In plants, the primary input of nitrogen (obtained from the soil or from symbiotic dinitrogen fixation) occurs through the assimilation of ammonia into organic form. Synthesis of glutamine (via glutamine synthetase) is the major, and possibly exclusive, route for this process, and there is little evidence for the participation of glutamate dehydrogenase. A variety of reactions distribute glutamine nitrogen to other compounds, including transfer to amino nitrogen through glutamate synthase. In many plants asparagine is a major recipient of glutamine nitrogen and provides a mobile reservoir for transport to sites of growth; ureides perform a similar function in some legumes. Utilisation of transport forms of nitrogen, and a number of other metabolic processes, involves release of ammonia, which must be reassimilated. In illuminated leaves, there is an extensive flux of ammonia released by the photorespiratory cycle, requiring continuous efficient reassimilation. Aspects of ammonia recycling and related amide metabolism in higher plants are reviewed.

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism

1981 ◽  
Vol 1 (2) ◽  
pp. 158-164
Author(s):  
N S Dunn-Coleman ◽  
E A Robey ◽  
A B Tomsett ◽  
R H Garrett
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Glutamate Synthase ◽  
Wild Type ◽  
Mutant Strains ◽  
Synthase Regulation ◽  
Glutamate Biosynthesis ◽  
Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

scholarly journals Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production

Forests ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1336
Author(s):  
Guizhi Fan ◽  
Tingting Zhang ◽  
Yingtian Liu ◽  
Yaguang Zhan ◽  
Baojiang Zheng
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Cell Cultures ◽  
Regulatory Mechanism ◽  
Glutamate Synthase ◽  
Suspension Cell ◽  
Suspension Cells ◽  
Synthetic Enzymes ◽  
Nos Inhibitor ◽  
Suspension Cell Cultures

Putrescine (Put) can enhance secondary metabolite production, but its intrinsic regulatory mechanism remains unclear. In this study, Put treatment promoted betulin production and gene expression of lupeol synthase (LUS), one of betulin synthetic enzymes. The maximum betulin content and gene expression level of LUS was 4.25 mg·g−1 DW and 8.25 at 12 h after 1 mmol·L−1 Put treatment, approximately two- and four-times that in the control, respectively. Put treatment increased the content of nitric oxide (NO) and its biosynthetic enzyme activity of nitrate reductase (NR) and NO synthase (NOS). Pretreatment of the birch suspension cells with NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO), NR inhibitor sodium azide (NaN3), and NOS inhibitor NG-nitro-L-Arg methyl ester (L-NAME) decreased Put-triggered NO generation and blocked Put-induced betulin production. Put treatment improved the content of NH4+ and its assimilation enzyme activity of glutamate synthase and glutamate dehydrogenase. NH4+ supplementation also promoted NO and betulin production. Thus, the above data indicated that Put-induced NO was essential for betulin production. NO derived from NR, NOS, and NH4+ mediated betulin production in birch suspension cell cultures under Put treatment.

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