Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain

被引:11
作者
Cha, J
Sorensen, MV
Ye, QZ
Auld, DS [1 ]
机构
[1] Harvard Univ, Sch Med, Ctr Biochem & Biophys Sci & Med, Boston, MA 02115 USA
[2] Brigham & Womens Hosp, Boston, MA 02115 USA
[3] Warner Lambert Parke Davis, Parke Davis Pharmaceut Res, Ann Arbor, MI 48105 USA
来源
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY | 1998年 / 3卷 / 04期
基金
美国国家卫生研究院;
关键词
matrix metalloproteinase; metal chelating agent; metal hybrid; fluorescent peptide substrate;
D O I
10.1007/s007750050244
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
We have selectively replaced the catalytic zinc of the catalytic domain of stromelysin-l (SCD) with other transition metals. Dialysis of the enzyme against 2 mM 1,10-phenanthroline, 20 mM Hepes, pH 7.5 in the presence of 10 mM CaCl2 removes the catalytic zinc, leaving the structural zinc site intact. Dialysis with metal-free buffer followed by the new metal ion replaces the catalytic zinc forming a metal hybrid enzyme. Full incorporation of 1 mol Co2+, Ni2+, or Cd2+/mol enzyme is confirmed by atomic absorption spectrometry while the weaker binding Mn2+ yields a value of 0.4 mol Mn2+/mol enzyme after dialysis against 1 mu M Mn2+. The activity of the monozinc enzyme is <10% while its activity is restored upon the addition of zinc and other transition metals. The k(cat) values for the Co2+, Mn2+, Cd2+, and Ni2+ enzymes are respectively 99%, 54%, 19%, and 17% of the value for the native enzyme, while the respective k(cat)/K-m values are 36%, 29%, 7%, and 16% toward the fluorescent heptapeptide substrate, DnsPLALRAR. The zinc and metal hybrid SCD cleave DnsPLA down arrow LRAR, and DnsPLE down arrow LFAR, exclusively at one bond, while DnsPLA down arrow L down arrow WAR and DnsPLA down arrow L down arrow FAR are cleaved at two positions. The double cleavage of DnsPLALWAR and DnsPLALFAR catalyzed by SCD is in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis is the different cleavage site specificity of the metal hybrids toward the A-L and L-W bonds of the DnsPLALWAR substrate. Thus the k(cat) values of: the Co/Zn hybrid for the cleavage of the A-L bond in the DnsPLALRAR and DnsPLAWAR substrates are 5- and X-fold greater than those for the Cd/Zn hybrid compared to a 140-fold difference for the corresponding k(cat) values for the L-W bond cleavage. These results imply that the catalytic metal of SCD is not only involved in catalysis but also influences the substrate specificity of the enzyme.
引用
收藏
页码:353 / 359
页数:7
相关论文
共 30 条
[1]   STROMELYSIN-1 - 3-DIMENSIONAL STRUCTURE OF THE INHIBITED CATALYTIC DOMAIN AND OF THE C-TRUNCATED PROENZYME [J].
BECKER, JW ;
MARCY, AI ;
ROKOSZ, LL ;
AXEL, MG ;
BURBAUM, JJ ;
FITZGERALD, PMD ;
CAMERON, PM ;
ESSER, CK ;
HAGMANN, WK ;
HERMES, JD ;
SPRINGER, JP .
PROTEIN SCIENCE, 1995, 4 (10) :1966-1976
[2]   Metal and pH dependence of heptapeptide catalysis by human matrilysin [J].
Cha, JH ;
Pedersen, MV ;
Auld, DS .
BIOCHEMISTRY, 1996, 35 (49) :15831-15838
[3]  
CLAVEL C, 1992, B CANCER, V79, P261
[4]   SYNTHESIS OF A FLUORESCENT DERIVATIZING REAGENT, 6-AMINOQUINOLYL-N-HYDROXYSUCCINIMIDYL CARBAMATE, AND ITS APPLICATION FOR THE ANALYSIS OF HYDROLYSATE AMINO-ACIDS VIA HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY [J].
COHEN, SA ;
MICHAUD, DP .
ANALYTICAL BIOCHEMISTRY, 1993, 211 (02) :279-287
[5]  
Gooley PR, 1996, J BIOMOL NMR, V7, P8
[6]   THE NMR STRUCTURE OF THE INHIBITED CATALYTIC DOMAIN OF HUMAN STROMELYSIN-1 [J].
GOOLEY, PR ;
OCONNELL, JF ;
MARCY, AI ;
CUCA, GC ;
SALOWE, SP ;
BUSH, BL ;
HERMES, JD ;
ESSER, CK ;
HAGMANN, WK ;
SPRINGER, JP ;
JOHNSON, BA .
NATURE STRUCTURAL BIOLOGY, 1994, 1 (02) :111-118
[7]   A SEMICONTINUOUS, HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-BASED ASSAY FOR STROMELYSIN [J].
HARRISON, R ;
TEAHAN, J ;
STEIN, R .
ANALYTICAL BIOCHEMISTRY, 1989, 180 (01) :110-113
[8]   MECHANISTIC STUDIES ON THE HUMAN MATRIX METALLOPROTEINASE STROMELYSIN [J].
HARRISON, RK ;
CHANG, B ;
NIEDZWIECKI, L ;
STEIN, RL .
BIOCHEMISTRY, 1992, 31 (44) :10757-10762
[9]   MATRIX METALLOPROTEINASE-7 (MATRILYSIN) FROM HUMAN RECTAL-CARCINOMA CELLS - ACTIVATION OF THE PRECURSOR, INTERACTION WITH OTHER MATRIX METALLOPROTEINASES AND ENZYMATIC-PROPERTIES [J].
IMAI, K ;
YOKOHAMA, Y ;
NAKANISHI, I ;
OHUCHI, E ;
FUJII, Y ;
NAKAI, N ;
OKADA, Y .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (12) :6691-6697
[10]   STROMELYSIN IS AN ACTIVATOR OF PROCOLLAGENASE - A STUDY WITH NATURAL AND RECOMBINANT ENZYMES [J].
MURPHY, G ;
COCKETT, MI ;
STEPHENS, PE ;
SMITH, BJ ;
DOCHERTY, AJP .
BIOCHEMICAL JOURNAL, 1987, 248 (01) :265-268