Shimotohno K W, Seto H, Otake N, Imai S, Murakami T
Institute of Applied Microbiology, University of Tokyo, Japan.
J Antibiot (Tokyo). 1988 Aug;41(8):1057-65. doi: 10.7164/antibiotics.41.1057.
2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.
2-膦酰甲基苹果酸（PMM）合酶催化草酰乙酸类似物膦酰丙酮酸（PPA）与乙酰辅酶A缩合形成PMM。该酶从双丙氨膦产生菌吸水链霉菌SF-1293的无细胞提取物中纯化了约700倍，达到电泳纯状态。通过SDS-聚丙烯酰胺凝胶电泳测定，纯化的PMM合酶亚基分子量为48,000，通过凝胶过滤法测定其天然分子量约为90,000至98,000。PMM合酶相对不稳定，在pH 8.0和30℃时表现出最大活性，并受到对氯汞苯甲酸、碘乙酰胺和EDTA的强烈抑制。EDTA抑制的酶活性通过添加Co++或Mn++可完全恢复，添加Ca++﹑Fe++或Mg++可部分恢复。该酶的特异性底物除了乙酰辅酶A外，还有PPA或草酰乙酸。在存在α-酮酸（如丙酮酸、α-酮戊二酸、脱氨基-α-酮去甲基膦丝菌素或膦酰丙酮酸）的情况下，该酶不催化乙酰辅酶A释放CoA。当使用丙酰辅酶A或丁酰辅酶A代替乙酰辅酶A作为底物时，缩合反应不发生。该酶对乙酰辅酶A的Km值为0.05 mM，对PPA为0.39 mM，对草酰乙酸为0.13 mM。PMM合酶在巯基化合物抑制模式、金属离子需求和立体特异性方面与梭菌的（R）-柠檬酸合酶非常相似；与（R）-柠檬酸合酶不同，PMM合酶不受氧气抑制。
