Volume 272, Number 3,
Issue of January 17, 1997
pp. 1444-1447
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
Cooperative Binding of Ca2+ to Human Interstitial
Collagenase Assessed by Circular Dichroism, Fluorescence, and Catalytic
Activity
(Received for publication, September 17, 1996, and in revised form, November 20, 1996)
Yan-na
Zhang
,
William L.
Dean
and
Robert D.
Gray
From the Department of Biochemistry, University of Louisville
School of Medicine, Louisville, Kentucky 40292
Dissociation of Ca2+ from human
interstitial collagenase induced either by chelation with EGTA or by
dilution resulted in loss of enzyme activity, a red shifted emission
maximum from 334 to 340 nm and quenching of protein fluorescence by
10% at 340 nm. Circular dichroism indicated that secondary structure
was unaffected by EGTA. Ca2+ binding to the EGTA-treated
enzyme as assessed by fluorescence was cooperative (Hill coefficient,
2.9; 50% saturation at 0.4 mM Ca2+). The
dependence of catalytic activity on [Ca2+] was also
cooperative (Hill coefficient, 1.7-2.0; midpoint [Ca2+],
0.2 mM). The Ca2+-reconstituted protein was
indistinguishable from the untreated enzyme by activity and
fluorescence measurements. These results demonstrate that removal of
Ca2+ from full-length collagenase generates a catalytically
incompetent, partially unfolded state with native secondary structure
but altered tertiary structure characterized by exposure of at least
one tryptophyl residue to a more polar environment.
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