The Catalytic Machinery of Chondroitinase ABC I Utilizes a Calcium Coordination Strategy to Optimally Process Dermatan Sulfate

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• 作者：Vikas Prabhakar ; Ishan Capila ; Rahul Raman ; Aravind Srinivasan ; Carlos J. Bosques ; Kevin Pojasek ; Michael A. Wrick ; Ram Sasisekharan
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：September 19, 2006
• 年：2006
• 卷：45
• 期：37
• 页码：11130 - 11139
• 全文大小：243K
• 年卷期：v.45,no.37(September 19, 2006)
• ISSN：1520-4995

文摘

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/ordermatan 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 providedimportant insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalention, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitinsubstrates in a concentration-dependent manner. Through biochemical and biophysical investigations, wehave established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calciumanalogue, confirm the specificity of this interaction. On the basis of theoretical structural models of theenzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordinationwere identified. These amino acids were investigated through site-directed mutagenesis studies and kineticassays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in theactive site of the enzyme.