Acetyl phosphate is known to phosphorylate several regulatory proteins of Escherichia coli and therefore was proposed as a global regulator. A number of proteins in crude extract were observed to be phosphorylated by acetyl phosphate in vitro, and six of them were characterized here. A sequencing of N-terminal amino acids revealed that they are phosphopentomutase, succinyl-CoA synthetase, phosphoglycerate mutase, nucleoside diphosphate kinase, and an unknown protein. Also included was CheY, a response regulator protein identified by immunoblotting. The chemical properties of the phosphoryl groups of the proteins indicated that acetyl phosphate may use the same residues, His or Asp, previously characterized as phosphorylation sites. Phosphorylation of the enzymes with acetyl phosphate followed slower kinetics than that of CheY, taking more than 5 min to reach their steady state levels of phosphorylation.
The role of acetyl phosphate in vivo was investigated by devising a method for in vivo phosphorylation using minicells. Labeling with inorganic [$^32P$]phosphate revealed that the phosphorylation levels of succinyl-CoA synthetase, phosphoglycerate mutase and CheY in minicells were higher in ackA mutant than in pta ackA double mutant, supporting in vivo phosphorylation by acetyl phosphate. These observations made on the enzymes which are implicated in the metabolic shift between biosynthesis and degradation led us to propose that acetyl phosphate serves as a metabolic modulator affecting the growth rate of cells. The role of acetyl phosphate in the reaction catalyzed by succinyl- CoA synthetase was investigated. The phosphorylation of succinyl-CoA synthetase alpha-subunit by acetyl phosphate requires the presence of magnesium. Chemical properties of the phosphoryl group and peptide mapping indicate that the phosphorylation site by acetyl phosphate would probably be the active site histidine residue. The enzyme utilizes acetyl phosphate and ADP to produce ATP, whereas synthesis of acetyl phosphate from ATP, the reverse reaction, was not favored. Consistent with this observation, acetyl phosphate was demonstrated to inhibit the activity of succinyl-CoA synthetase by competing with ATP but not with succinate, leading to a model for the reaction by acetyl phosphate.
Some of the phosphorylated proteins observed by labeling the minicells with inorganic [$^32P$]phosphate were characterized. It was found that an addition of glucose or methyl alpha-glucoside resulted in dephosphorylation of some proteins, not phosphorylated in the minicells from ptsI mutants, indicating that they are components of the phosphotransferase system. The chemical properties of their phosphoryl groups indicated that they are all phosphohistidines except for one, which was extremely unstable and thus likely to be an acyl-phosphate. One of the proteins whose state of phosphorylation were dependent on glucose was identified as $IIA^Glc$ by comparison of its mobility on polyacrylamide gel elctrophoresis with that of $IIA^Glc$ band in crude extract. Moreover, its steady-state level of phosphorylation was found to be dependent on the concentration of external glucose. This result supports the model that $IIA^Glc$ functions as a key regulatory molecule in inducer exclusion or activation of adenylate cyclase by changing its phosphorylation level in response to an external glucose concentration.
Acetyl phosphate는 대장균의 여러 가지 조절 단백질 (regulatory protein)을 인산화시키는 것으로 알려져 있고, global regulator로서의 역할이 제시된 바 있다. 본 연구에서는 조효소의 많은 단백질들이 in vitro에서 acetyl phosphate에 의해 인사화되는 것을 관찰하였고, 그 중 6개의 특성을 조사하였다. N-말단의 아미노산배열로서 그것들이 phosphopentomutase, succinyl-CoA synthetase, phosphoglycerate을 밝혔고, immunoblotting으로 다른 하나는 반응조절자 (response regulator)인 CheY임을 알았다. 단백질의 인산기의 화학적 특성을 조사한 결과, 이미 인사화되는 위치로 알려진 His 또는 Asp 잔기가 acetyl phosphate에 의해 인산화 되는 것으로 추측되었다. Acetyl phosphate에 의한 효소들의 인산화 속도는 CheY보다 느려서 인산화의 steady state phosphate의 역할을 조사하기 위하여 minicell을 이용한 in vivo 인산화 방법을 고안하였다. Minicell을 $[^{32}P]$무기인산으로 표지한 결과, pta ackA 돌연변이주보다 ackA 돌연변이주에서 syccinyl-CoA synthetase, phosphoglycerate mutase, 그리고 CheY의 인산화 수준이 더 높았고, 이는 acetyl phosphate에 의한 in vivo 인산화를 뒷받침하는 결과이다. Acetyl phosphate에 의해 인산화되는 효소들이 대사과정에서 생합성과 분해의 분기점에 있다는 사실과 이상의 결과로부터 균의 성장속도를 조절하는 metabolic modulator로서의 acetyl phosphate의 역할을 제시하였다.
Succinyl-CoA synthetase에 의해 촉매되는 반응에서의 acetyl phosphate의 역할을 조사하였다. Acetyl phosphate에 의한 succinyl-CoA synthetase의 α-subunit의 인산화에는 magnesium이 필요하였고, 인산기의 화학적 특성과 peptide mapping으로 acetyl phosphate에 의한 인산화 위치는 active site histidine 잔기임을 알았다. Succinyl-CoA synthetase는 acetyl phosphate와 ADP를 기질로하여 ATP를 생성하였으나, 반대방향의 반응, 즉 ATP로부터 acetyl phosphate의 생성은 잘 일어나지 않았다. 한편, acetyl phosphate는 ATP와 competition하고 succinate와는 competition하지 않으면서 succinyl-CoA synthetase 활성을 저해하였고, 이를 바탕으로 acetyl phosphate에 의한 반응 모델을 제시하였다.
Minicell을 $[^{32}P]$무기인산으로 표지하였을 때 관찰되는 인산화 단백질의 특성을 조사하였다. Glucose나 methyl α-glucoside를 첨가하였을 때 인산화가 감소하는 단백질들은 ptsI 돌연변이주로부터 얻은 minicell에서는 인산화되지 않는 것을 발견하였고, 이는 이 단백질들이 phosphotransferase system의 component라는 것을 의미한다. 이 단백질들의 인산기는, 매우 불안정하여 acyl-phosphate라고 추측되는 하나를 제외하고는, 모두 phosphohistidine임을 화학적 특성으로부터 알았다. 이 단백질들중 하나는 전기영동상의 이동성으로 $IIA^{Glc}$ 임이 밝혀졌고, 그것의 인산화 수준은 세포밖의 glucose 농도에 의해 결정되는 것을 관찰하였다. 이 결과는 $IIA^{Glc}$가 세포밖의 Glucose 농도에 따라 인산화 수준을 변화하여 inducer exculsion이나 adenylate cyclase의 활성을 조절하는 조절자의 역할을 한다는 모델을 뒷받침한다.
