Hager P W, Collart F R, Huberman E, Mitchell B S
Department of Pharmacology, University of North Carolina at Chapel Hill 27514, USA.
Biochem Pharmacol. 1995 May 11;49(9):1323-9. doi: 10.1016/0006-2952(95)00026-v.
Inosine monophosphate dehydrogenase (IMPDH) activity results from the expression of two separate genes, and the resulting proteins (type I and type II) are 84% identical at the amino acid level. Although the type II mRNA is expressed at higher levels in proliferating cells, both mRNAs, and by extrapolation both proteins, are present in normal and malignant cells. Since IMPDH is an important target for the development of drugs with both chemotherapeutic and immunosuppressive activity, we have compared the kinetic and physical properties of the two human enzymes expressed in and purified from Escherichia coli. Type I and II IMPDH had kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5'-monophosphate (MMP) with Ki values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with Ki values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with kon values of 3-8 x 10(4) M-1 sec-1 and koff values of 3 x 10(-4) sec-1 (half-lives of 36-43 min). Thus, MMP is a potent, tight-binding competitive inhibitor that does not discriminate between the two IMPDH isozymes.
肌苷单磷酸脱氢酶（IMPDH）活性源于两个独立基因的表达，所产生的蛋白质（I型和II型）在氨基酸水平上有84%的同一性。尽管II型mRNA在增殖细胞中表达水平较高，但两种mRNA，由此推断两种蛋白质，在正常细胞和恶性细胞中均有存在。由于IMPDH是开发具有化疗和免疫抑制活性药物的重要靶点，我们比较了在大肠杆菌中表达并纯化的两种人源酶的动力学和物理性质。I型和II型IMPDH的kcat值分别为1.8和1.4秒-1，IMP的Km值分别为14和9微摩尔，NAD的Km值分别为42和32微摩尔。这两种酶被免疫抑制剂米唑立宾5'-单磷酸（MMP）竞争性抑制，Ki值分别为8和4纳摩尔，被霉酚酸非竞争性抑制，Ki值分别为11和6纳摩尔。通过荧光猝灭监测，MMP与任一同工酶的结合相对较慢，kon值为3 - 8×10(4) M-1秒-1，koff值为3×10(-4)秒-1（半衰期为36 - 43分钟）。因此，MMP是一种强效、紧密结合的竞争性抑制剂，对两种IMPDH同工酶没有区分性。
