Suda S A, Dolmer K, Gettins P G
Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60612-4316, USA.
J Biol Chem. 1997 Dec 5;272(49):31107-12. doi: 10.1074/jbc.272.49.31107.
It has been shown that the relative reaction preference of the C4 thiol ester toward oxygen and nitrogen nucleophiles upon activation by proteinase depends on whether residue 1106 is aspartate or histidine (Dodds, A. W., Ren, X.-D., Willis, A. C., and Law, S. K. A. (1996) Nature 379, 177-179). To determine if the equivalent residue in the related thiol ester-containing protein human alpha2-macroglobulin (alpha2M), asparagine 1065, plays a similar role, we have expressed and characterized four alpha2M variants in which this asparagine has been replaced by aspartate, alanine, histidine, or lysine. The change from asparagine resulted in an altered ability to form the thiol ester. This ranged from failure to form the thiol ester (Asn --> Asp) to a maximum extent of formation of about 50% (Asn --> Ala). For the three variants that were able to form the thiol ester, the rates of thiol ester cleavage by a given amine were found to be different from one another and slower in nearly all cases than plasma alpha2M, but with the same relative reactivity of methylamine > ethylamine > ammonia. The rate of conformational change that follows cleavage of thiol esters in a functional half-molecule was also found to differ between the variants and to be slower than plasma alpha2M. TNS emission spectra indicated that the conformations of the transformed variants differed measurably from transformed plasma alpha2M. These findings suggest that residue 1065 plays a critical role in human alpha2M, for formation of the thiol ester, for its subsequent reaction with nucleophiles, and for the conformational change induced by this reaction. By analogy with C4, where this residue influences the nucleophile preference through direct interaction with the thiol ester, residue 1065 in alpha2M is expected to be located in or very close to the thiol ester region in alpha2M.
研究表明，蛋白酶激活后，C4硫酯对氧亲核试剂和氮亲核试剂的相对反应偏好取决于1106位残基是天冬氨酸还是组氨酸（多兹，A.W.，任，X.-D.，威利斯，A.C.，以及劳，S.K.A.（1996年）《自然》379卷，177 - 179页）。为了确定相关的含硫酯蛋白人α2 - 巨球蛋白（α2M）中的等效残基天冬酰胺1065是否起类似作用，我们表达并表征了四种α2M变体，其中该天冬酰胺已被天冬氨酸、丙氨酸、组氨酸或赖氨酸取代。从天冬酰胺的改变导致形成硫酯的能力发生变化。这一变化范围从无法形成硫酯（天冬酰胺→天冬氨酸）到形成程度最大约为50%（天冬酰胺→丙氨酸）。对于能够形成硫酯的三种变体，发现给定胺对硫酯的裂解速率彼此不同，并且在几乎所有情况下都比血浆α2M慢，但甲胺＞乙胺＞氨的相对反应性相同。在功能性半分子中硫酯裂解后伴随发生的构象变化速率在变体之间也有所不同，并且比血浆α2M慢。TNS发射光谱表明，转化变体的构象与转化血浆α2M有明显差异。这些发现表明，残基1065在人α2M中对于硫酯的形成、其随后与亲核试剂的反应以及该反应诱导的构象变化起着关键作用。与C4类似，在C4中该残基通过与硫酯的直接相互作用影响亲核试剂偏好，预计α2M中的残基1065位于α2M的硫酯区域内或非常靠近该区域。
