Kovachy R J, Copley S D, Allen R H
J Biol Chem. 1983 Sep 25;258(18):11415-21.
Certain amino acids and other compounds are metabolized via: Propionyl-CoA carboxylase in equilibrium D-methylmalonyl-CoA racemase in equilibrium L-methylmalonyl-CoA (adenosylcobalamin) mutase in equilibrium succinyl-CoA in equilibrium tricarboxylic acid cycle. In cobalamin deficiency and in genetic disorders involving adenosylcobalamin or the mutase, large amounts of methylmalonic acid are excreted in the urine. Its origin is unknown, however, since nonesterified methylmalonic acid is not present in the above or other known pathways. To investigate the origin of methylmalonic acid, we fractionated rat liver by gel filtration and found a single peak (Mr = 35,000) of activity for the hydrolysis of DL-methylmalonyl-CoA to methylmalonic acid and CoA. The enzyme has been purified 3,100-fold with a yield of 2.1% from 1.6 kg of rat liver using a purification scheme consisting of ammonium sulfate fractionation, ion exchange chromatography on CM (carboxymethyl)-cellulose and DEAE-cellulose, affinity chromatography on CoA-agarose, and gel filtration on Sephadex G-100. The final preparation gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme is 35,000 based on gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis in the presence and absence of 1% 2-mercaptoethanol. The enzyme was shown to be active on the D-isomer, but not on the L-isomer, of methylmalonyl-CoA by CD spectropolarimetry. The Km for D-methylmalonyl-CoA is 0.7 mM and the molar activity is 1,400 molecules of substrate/min/molecule of enzyme. At substrate concentrations of 0.5 mM, the relative rate of hydrolysis of CoA esters was as follows: D-methylmalonyl-CoA (100%), malonyl-CoA (16%), propionyl-CoA (3%), acetyl-CoA (1%), succinyl-CoA (less than 1%), and palmitoyl-CoA (less than 1%). This enzyme appears to account for the markedly increased amounts of methylmalonic acid that are excreted in the urine in cobalamin deficiency and in genetic disorders involving adenosylcobalamin or L-methylmalonyl-CoA mutase.
丙酰辅酶A羧化酶催化反应生成平衡态的D - 甲基丙二酰辅酶A，D - 甲基丙二酰辅酶A消旋酶催化其生成平衡态的L - 甲基丙二酰辅酶A，L - 甲基丙二酰辅酶A（腺苷钴胺素）变位酶催化其生成平衡态的琥珀酰辅酶A，琥珀酰辅酶A进入三羧酸循环。在钴胺素缺乏以及涉及腺苷钴胺素或变位酶的遗传性疾病中，大量甲基丙二酸经尿液排出。然而，其来源尚不清楚，因为上述或其他已知途径中不存在未酯化的甲基丙二酸。为了研究甲基丙二酸的来源，我们通过凝胶过滤对大鼠肝脏进行分级分离，发现了一个单一的活性峰（Mr = 35,000），该活性峰可将DL - 甲基丙二酰辅酶A水解为甲基丙二酸和辅酶A。使用由硫酸铵分级分离、CM（羧甲基） - 纤维素和DEAE - 纤维素离子交换色谱、辅酶A - 琼脂糖亲和色谱以及Sephadex G - 100凝胶过滤组成的纯化方案，从1.6千克大鼠肝脏中纯化该酶，纯化倍数达到3100倍，产率为2.1%。最终制剂在聚丙烯酰胺凝胶电泳上呈现单一条带。基于凝胶过滤以及在有和没有1% 2 - 巯基乙醇存在下的十二烷基硫酸钠 - 聚丙烯酰胺电泳，纯化酶的分子量为35,000。通过圆二色光谱偏振法表明该酶对甲基丙二酰辅酶A的D - 异构体有活性，但对L - 异构体无活性。D - 甲基丙二酰辅酶A的Km为0.7 mM，摩尔活性为1400个底物分子/分钟/酶分子。在底物浓度为0.5 mM时，辅酶A酯的相对水解速率如下：D - 甲基丙二酰辅酶A（100%）、丙二酰辅酶A（16%）、丙酰辅酶A（3%）、乙酰辅酶A（1%）、琥珀酰辅酶A（小于1%）和棕榈酰辅酶A（小于1%）。这种酶似乎可以解释在钴胺素缺乏以及涉及腺苷钴胺素或L - 甲基丙二酰辅酶A变位酶的遗传性疾病中，经尿液排出的甲基丙二酸量显著增加的现象。
