Clarke L, Waxman D J
Arch Biochem Biophys. 1987 Aug 1;256(2):585-96. doi: 10.1016/0003-9861(87)90616-3.
Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.
通过0 - 30%（w/v）硫酸铵分级分离从人肝细胞溶质中分离出叶酰聚谷氨酸合成酶（FPGS），并对其进行了生化特性分析。以氨基蝶呤（AMT）、L - [³H]谷氨酸和MgATP作为共底物，在激活剂KCl和NaHCO₃存在的情况下观察到最大的γ - L - 谷氨酰化活性。ATP和2 - 巯基乙醇对于酶活性和稳定性均是必需的。在没有ATP的情况下，人肝FPGS在37℃迅速失活（半衰期约8分钟），而从兔肝中分离的FPGS稳定性显著更高（半衰期 = 68分钟）。分离出的人肝酶能有效地将叶酸和抗叶酸药物聚谷氨酸化。测定的AMT的Km参数（Km = 4.3 μM）与几种还原型叶酸（四氢叶酸、二氢叶酸和亚叶酸；Km = 3 - 7 μM）测定的参数相似，而甲氨蝶呤（MTX）、5 - 甲基四氢叶酸（Km = 50 - 60 μM）和叶酸（Km = 100 μM）的Km值显著更高。所有检测的底物的Vmax值范围为AMT值的30%至90%（Vmax = 935 pmol产物/毫克/小时）。这些底物的反应活性顺序与从大鼠和兔肝中分离的FPGS的平行研究中确定的顺序不同。就AMT和几种还原型叶酸而言，在底物浓度达到或高于50 - 250 μM时观察到对人肝FPGS的抑制作用。从六个个体的人肝中分离出的FPGS表现出高度相似的生化和动力学特性，表明每个个体中存在相同或至少高度相似的酶种类，观察到的比活性个体间范围为五倍。将MTX与其更高聚谷氨酸化产物（MTX - Glu2至MTX - Glu6）作为FPGS底物进行比较，结果表明随着谷氨酸链长度增加，Vmax值显著降低，这部分被Km的相应降低所补偿。与这些观察结果一致，当以MTX作为底物时，分离出的酶无法合成高于MTX - Glu3的聚谷氨酸，这就引发了一个问题，即体内如何形成含有多达五个或六个γ - L - 谷氨酸残基的MTX聚谷氨酸。
