Critical role of asparagine 1065 of human alpha2-macroglobulin in formation and reactivity of the thiol ester.

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1065, plays a similar role, we have expressed and characterized four alpha2M variants in which this asparagine has been replaced by aspartate, alanine, histidine, or lysine.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.