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 .times. 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.