The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate.
The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.
Prabhakar
Vikas
V
Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Capila
Ishan
I
Raman
Rahul
R
Srinivasan
Aravind
A
Bosques
Carlos J
CJ
Pojasek
Kevin
K
Wrick
Michael A
MA
Sasisekharan
Ram
R
D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D002413:Cations, Divalent; D003871:Dermatan Sulfate; D019765:Chondroitin ABC Lyase; D002118:Calcium