High-Affinity Calcium-Binding Site in the γ-Carboxyglutamic Acid Domain of Bovine Factor VII†

Biochemistry ◽  
1996 ◽  
Vol 35 (43) ◽  
pp. 13826-13832 ◽  
Author(s):  
Keisuke Inoue ◽  
Hidenori Shimada ◽  
Junichi Ueba ◽  
Satoru Enomoto ◽  
Yukari Tanaka-Saisaka ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Factor Vii ◽  
Calcium Binding Site ◽  
High Affinity ◽  
Carboxyglutamic Acid ◽  
Acid Domain ◽  
Bovine Factor

scholarly journals Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

2007 ◽  
Vol 403 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Seakwoo Lee ◽  
Hyun I. Park ◽  
Qing-Xiang Amy Sang
Keyword(s):  
Breast Cancer ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Calcium Binding ◽  
Human Breast Cancer ◽  
Structural Changes ◽  
Human Breast Cancer Cell ◽  
Human Breast ◽  
Calcium Binding Site ◽  
High Affinity

Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a Kd2 (low-affinity calcium-dissociation constant) value of 120 μM, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

1990 ◽  
Vol 9 (2) ◽  
pp. 475-480 ◽  
Author(s):  
P.A. Handford ◽  
M. Baron ◽  
M. Mayhew ◽  
A. Willis ◽  
T. Beesley ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Human Factor ◽  
Factor Ix ◽  
Calcium Binding Site ◽  
High Affinity ◽  
Human Factor Ix

Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin

Biochemistry ◽  
2013 ◽  
Vol 52 (50) ◽  
pp. 9080-9088 ◽  
Author(s):  
Ryo Uehara ◽  
Clement Angkawidjaja ◽  
Yuichi Koga ◽  
Shigenori Kanaya
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Insertion Sequence ◽  
Calcium Binding Site ◽  
High Affinity ◽  
Subtilisin E

A functional calcium-binding site in the metalloprotease domain of ADAMTS13

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1149-1157 ◽  
Author(s):  
Michelle D. Gardner ◽  
Chan K. N. K. Chion ◽  
Rens de Groot ◽  
Anuja Shah ◽  
James T. B. Crawley ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Calcium Binding ◽  
Major Influence ◽  
Calcium Binding Site ◽  
High Affinity ◽  
Fold Reduction ◽  
Reported Study ◽  
Specificity Constant ◽  
Von Willebrand

Abstract ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with KD(app) of 80 μM (± 15 μM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the KD(app) for Ca2+ of ADAMTS13, and for D187A and E212A also reduced the Vmax to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed an approximately 13-fold reduction in specificity constant, kcat/Km, contributed by changes in both Km and kcat. These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

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