1. Inhibition of homocysteine sulfonamide of glutamate synthase purified from Saccharomyces cerevisiae
D S Masters Jr, A Meister J Biol Chem. 1982 Aug 10;257(15):8711-5.
Glutamate synthase, isolated in apparently homogeneous form (Mr approximately 265,000) from Saccharomyces cerevisiae after 7500-fold purification, is markedly inhibited by homocysteine sulfonamide. Inhibitions competitive with respect to L-glutamine; the apparent Ki value calculated for L-homocysteine sulfonamide is 3.6 microM; the apparent Km value for L-glutamine is 280 microM. The very high affinity of the inhibitor for the enzyme, as well as structural considerations, suggest that homocysteine sulfonamide is a transition state inhibitor. The previously reported growth inhibitory properties of homocysteine sulfonamide (Reisner, D. B. (1958) J. Am. Chem. soc. 78, 5102-5104) may be due, at least in part, to inhibition of glutamate synthase. L-Methionine sulfone is also a potent competitive inhibitor, whereas L-albizziin, L-methionine-SR-sulfoximine, and L-methionine-SR-sulfoxide are much less effective inhibitors. S. cerevisiae glutamate synthase, which is composed of two dissimilar subunits, uses NADH exclusively, and exhibits low but definite activity when NH3 is substituted for glutamine.
2. Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs
H Chmara, R Andruszkiewicz, E Borowski Biochim Biophys Acta. 1986 Mar 28;870(2):357-66. doi: 10.1016/0167-4838(86)90240-2.
A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.
3. Regulation of assimilatory nitrate reductase activity in soil by microbial assimilation of ammonium
G W McCarty, J M Bremner Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):453-6. doi: 10.1073/pnas.89.2.453.
It is well established that assimilatory nitrate reductase (ANR) activity in soil is inhibited by ammonium (NH4+). To elucidate the mechanism of this inhibition, we studied the effect of L-methionine sulfoximine (MSX), an inhibitor of NH4+ assimilation by microorganisms, on assimilatory reduction of nitrate (NO3-) in aerated soil slurries treated with NH4+. We found that NH4+ strongly inhibited ANR activity in these slurries and that MSX eliminated this inhibition. We also found that MSX induced dissimilatory reduction of NO3- to NH4+ in soil and that the NH4+ thus formed had no effect on the rate of NO-3 reduction. We concluded from these observations that the inhibition of ANR activity by NH4+ is due not to NH4+ per se but to products formed by microbial assimilation of NH4+. This conclusion was supported by a study of the effects of early products of NH4+ assimilation (L amino acids) on ANR activity in soil, because this study showed that the biologically active, L isomers of glutamine and asparagine strongly inhibited ANR activity, whereas the D isomers of these amino acids had little effect on ANR activity. Evidence that ANR activity is regulated by the glutamine formed by NH4+ assimilation was provided by studies showing that inhibitors of glutamine metabolism (azaserine, albizziin, and aminooxyacetate) inhibited ANR activity in soil treated with NO3- but did not do so in the presence of MSX.
