Taylor A B, Johnson W H, Czerwinski R M, Li H S, Hackert M L, Whitman C P
Department of Chemistry and Biochemistry, Medicinal Chemistry Division, College of Pharmacy, The University of Texas, Austin 78712-1071, USA.
Biochemistry. 1999 Jun 8;38(23):7444-52. doi: 10.1021/bi9904048.
Macrophage migration inhibitory factor (MIF) exhibits dual activities. It acts as an immunoregulatory protein as well as a phenylpyruvate tautomerase. To understand better the relationship between these two activities and to elucidate the structural basis for the enzymatic activity, a crystal structure of a complex between murine MIF and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, has been determined to 1.8 A resolution. The structure is nearly superimposable on that of the free protein indicating that the presence of the inhibitor does not result in any major structural changes. The inhibitor also confirms the location of the active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adjacent subunits. At the back of the cavity, the side-chain carbonyl oxygen of Asn-97' interacts with the phenolic hydroxyl group of the inhibitor while at the mouth of the cavity the ammonium group of Lys-32 interacts with a carboxylate oxygen. The other carboxylate oxygen of the inhibitor interacts with Pro-1. The hydroxyl group of Tyr-95' interacts weakly with the fluoro group on the inhibitor. The hydrophobic side chains of five active-site residues (Met-2, Ile-64, Met-101, Val-106, and Phe-113) and the phenyl moiety of Tyr-95' are responsible for the binding of the phenyl group. Further insight into the enzymatic activity of MIF was obtained by carrying out kinetic studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyruvate. The results demonstrate that MIF processes the enol isomers more efficiently than the keto isomers primarily because of a decrease in Km. On the basis of these results, a mechanism is proposed for the MIF-catalyzed tautomerization reaction.
巨噬细胞移动抑制因子（MIF）具有双重活性。它作为一种免疫调节蛋白，同时也是一种苯丙酮酸互变异构酶。为了更好地理解这两种活性之间的关系，并阐明酶活性的结构基础，已确定了小鼠MIF与互变异构酶活性的竞争性抑制剂（E）-2-氟-对羟基肉桂酸之间复合物的晶体结构，分辨率为1.8埃。该结构与游离蛋白的结构几乎完全重叠，表明抑制剂的存在不会导致任何重大结构变化。抑制剂还确定了活性位点位于含有氨基末端脯氨酸的疏水腔内。在这个腔内，抑制剂与相邻亚基的残基相互作用。在腔的后部，Asn-97'的侧链羰基氧与抑制剂的酚羟基相互作用，而在腔口，Lys-32的铵基与羧酸根氧相互作用。抑制剂的另一个羧酸根氧与Pro-1相互作用。Tyr-95'的羟基与抑制剂上的氟基团弱相互作用。五个活性位点残基（Met-2、Ile-64、Met-101、Val-106和Phe-113）的疏水侧链以及Tyr-95'的苯基部分负责苯基的结合。通过使用苯丙酮酸和（对羟基苯基）丙酮酸的烯醇异构体进行动力学研究，对MIF的酶活性有了进一步了解。结果表明，MIF处理烯醇异构体比酮异构体更有效，主要是因为Km降低。基于这些结果，提出了MIF催化互变异构反应的机制。
