Langston-Unkefer P J, Robinson A C, Knight T J, Durbin R D
J Biol Chem. 1987 Feb 5;262(4):1608-13.
Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.
植物谷氨酰胺合成酶是丁香假单胞菌几种致病致病变种产生的毒素β-内酰胺tabtoxinine的生理靶点。这种毒素是一种独特的氨基酸，是豌豆谷氨酰胺合成酶的活性位点定向不可逆抑制剂。失活需要ATP。ADP、AMP或腺苷5'-（β，γ-亚甲基）三磷酸（AMP-PCP）均不能支持失活。通过31P NMR光谱分析确定，腺苷-5'-基亚磷酰亚胺（AMP-PNP）被谷氨酰胺合成酶缓慢水解，产生腺苷-5'-基磷酰胺（AMP-PN）和无机磷酸。AMP-PNP也支持β-内酰胺tabtoxinine对谷氨酰胺合成酶的缓慢失活。这些数据与γ-磷酸转移参与失活一致。完全失活的谷氨酰胺合成酶每亚基结合0.9 μmol毒素。在不存在毒素或另一种活性位点定向抑制剂甲硫氨酸亚砜亚胺的情况下，每亚基谷氨酰胺合成酶结合1 μmol ATP；然而，当谷氨酰胺合成酶在毒素或甲硫氨酸亚砜亚胺存在下孵育直至所有酶活性丧失时，每亚基结合第二μmol [α-或γ-32P]ATP。这些数据表明，失活过程中从ATP水解的γ-磷酸以及ADP仍与酶-抑制剂复合物结合。开链形式的tabtoxinine既不是谷氨酰胺合成酶的可逆抑制剂也不是不可逆抑制剂，这表明β-内酰胺环是抑制所必需的。在含有15%（v/v）乙二醇的缓冲液中，用β-内酰胺tabtoxinine使谷氨酰胺合成酶失活是准一级反应。该反应的速率常数为3×10-2 S-1，毒素的Ki为1 mM。从缓冲液中除去乙二醇后，反应以非一级方式进行，表观速率常数随时间降低。在这些条件下，随着酶失活，对毒素的结合亲和力似乎降低，而观察到的谷氨酸Km不变。
