Substrate variability as a factor in enzyme inhibitor design: inhibition of ovine brain glutamine synthetase by .alpha.- and .gamma.-substituted phosphinothricins

Abstract

Ovine brain glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid], a naturally occurring inhibitor of GS. These compounds display competitive inhibition of GS, and a correlation between the inhibitor Ki values and the Km/Vmax values of the analogously substituted glutamates supports the hypothesis that the phosphinothricins participate in transition-state analogue inhibition of GS. At concentrations greater than Ki these inhibitors caused biphasic time-dependent loss of enzyme activity, with initial pseudo-first-order behavior; k'inact parameters were determined for several compounds and were similar to the 2.1 X 10(-2)s-1 value measured for PPT. Dilution after GS inactivation caused a non-first-order recovery of activity. Reactivation kinetics were insensitive to inhibitor and ADP concentrations over wide ranges, although very high postdilution concentrations of inhibitor suppressed reactivation. The burst activity level, beta, as well as the concentration of inhibitor required to suppress reactivation to this level, mu, expressed as a multiple of the Ki value, was characteristic for each compound in the phosphinothricin series. Increasing substitution of the phosphinothricin parent structure caused an increase in Ki values as well as in the inactivation/reactivation parameters. The kinetic behavior of these inhibitors is consistent with a mechanistic scheme involving initial phosphorylation and rapid partial inhibitor dissociation, followed by slow release of remaining bound inhibitor.

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

“…[23][24][25][26][27] However, none of the 2-amino-phosphono-carboxylic acids (16)(17)(18) identified in the virtual screening showed any inhibitory activity, despite the fact that the 9 phosphono group of docked 2-amino-6-phosphonohexanoic acid (17) overlaps well with the phosphono group of MSO-P. This is in accordance with results for DL-homophosphinothricin, which did not show any inhibition of sheep brain GS.…”

“…This is in accordance with results for DL-homophosphinothricin, which did not show any inhibition of sheep brain GS. 23 Thus the patterns of inhibition observed here agree well between the prokaryotic and eukaryotic GSs.…”

“…In fact a number of inhibitors, including MSO, 8 or its diastereomeric mixture L-(SR), 8,11,13,23 PPT, 8,19,20,[22][23][24][25]28 3, 19,20 and 4 29,35 have shown inhibitory activity when tested on both bacterial and eukaryotic GSs. Therefore a literature search was performed to identify known GS inhibitors for both eukaryotic and prokaryotic enzymes.…”

“…[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] Much effort has been invested in developing GS inhibitors not only for antibacterial research, but also for weed control, 20,25,[30][31][32] since GS is a key enzyme in ammonia assimilation in plants. However, no inhibitor has shown improved activity compared to MSO or PPT.…”

The Metabolites of the Herbicide L-Phosphinothricin (Glufosinate) (Identification, Stability, and Mobility in Transgenic, Herbicide-Resistant, and Untransformed Plants)

“…The toxicology of t-Pt has been intensively analyzed (Lejczak et al, 1981;Lacoste et al, 1985;Dom et al, 1986;Logusch et al, 1989) and was summarized by Ebert et al (1990). It is safe under the conditions of recommended use, but, according to European Economic Community directive 83/467/EEC, it has to be classified as harmful on the basis of testing for acute oral toxicity (Ebert et al, 1990).…”