Characterization of the role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitory factor
Characterization of the role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitory factor
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DOI：
10.1021/bi9806955
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Stamps, SL;Fitzgerald, MC;Whitman, CP
细胞因子巨噬细胞移动抑制因子（MIF）通过未知或知之甚少的机制介导几种免疫和炎症过程。该蛋白与两种细菌异构酶4-羟巴豆酸互变异构酶（4-OT）和5-（羧甲基）-2-羟基粘康酸异构酶（CHMI）具有结构同源性，并催化苯丙酮酸的烯醇化和（对羟基苯基）丙酮酸的酮化。氨基末端的脯氨酸已被确定为催化碱在4-OT-和CHMI-催化的反应。MIF还具有氨基末端脯氨酸，其在MIF催化的反应中被认为是催化基团。为了进一步描述Pro-1在MIF催化反应中的作用，用3-溴丙酮酸（3-BP）进行亲和标记研究。研究结果表明，3-BP作为酶活性的活性位点定向的不可逆抑制剂，并且修饰每个单体亚基的一个位点。该抑制剂作为其乳酰衍生物共价连接到11个残基的氨基末端片段Pro-1至Arg-11。该肽片段内唯一合理的烷基化位点是氨基末端脯氨酸。因为对于k（inact）/K-I的pH依赖性测量的pK（a）（5.7 +/-0.2）和对于苯丙酮酸烯醇化的k（cat）/K-m的pH依赖性测量的pK（a）（6.0 +/-0.1）与通过其直接滴定获得的Pro-1的先前测量的pK（a）（5.6 +/-0.1）相当并且合理地一致[Swope，M.，Sun H.- W.，Blake，P.，和Lolls，E.（1998）EMBO J.（出版中）]，得出的结论是Pro-1在MIF催化的反应中充当一般的碱催化剂。结构和机理的相似之处在于，4-OT、CHMI和MIF属于一个超家族的酶，这些酶通过它们催化乙酰基部分的酮-烯醇互变异构化的能力而相关。
The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit, The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-ll. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pK(a) measured for the pH dependence of k(inact)/K-I (5.7 +/- 0.2) and that measured for the pH dependence of the k(cat)/K-m for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pK(a) of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolls, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The structural and mechanistic parallels place 4-OT, CHMI, and MIF in a superfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.