CHARACTERIZATION OF ZINC-BINDING SITES IN HUMAN STROMELYSIN-1 - STOICHIOMETRY OF THE CATALYTIC DOMAIN AND IDENTIFICATION OF A CYSTEINE LIGAND IN THE PROENZYME

被引:74
作者
SALOWE, SP [1 ]
MARCY, AI [1 ]
CUCA, GC [1 ]
SMITH, CK [1 ]
KOPKA, IE [1 ]
HAGMANN, WK [1 ]
HERMES, JD [1 ]
机构
[1] MERCK SHARP & DOHME LTD,DEPT MED CHEM RES,RAHWAY,NJ 07065
关键词
D O I
10.1021/bi00134a001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by > 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site. This spectroscopic evidence supports a model proposed for all matrix metalloproteinase proenzymes in which a zinc-cysteine interaction is required for the preservation of latency [Van Wart, H. E., & Birkedal-Hansen, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5578-5582].
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页码:4535 / 4540
页数:6
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