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Article

Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate^†

Eva Muñoz,^‡ Ding Xu,^§ Melissa Kemp,^‡ Fuming Zhang,^‡ Jian Liu,^§ and Robert J. Linhardt*^‡

Departments of Chemistry and Chemical Biology, Biology, and Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, and Department of Medicinal Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599

Biochemistry, 2006, 45 (16), pp 5122–5128

DOI: 10.1021/bi052403n

Publication Date (Web): March 30, 2006

†

This work is supported in part by NIH Grants HL52622, HL62244, and GM38060 (to R.J.L.) and AI 50050 (to J.L.). D.X. is a recipient of a predoctoral fellowship from the American Heart Association MidAtlantic Affiliate.

‡

Rensselaer Polytechnic Institute.

University of North Carolina.

To whom correspondence should be addressed. Phone: (518) 276-3404. Fax: (518) 276-3405. E-mail: linhar@rpi.edu.

Abstract

The 3-O-sulfonation of glucosamine residues in heparan sulfate (HS) by 3-O-sulfotransferase (3-OST) is a key substitution that is present in HS sequences of biological importance, in particular HS anticoagulant activity. Six different isoforms of 3-OST have been identified that exhibit different substrate specificity. In this paper the affinity and kinetics of the interaction between 3-O-sulfotransferase isoform 1 (3-OST-1) and HS have been examined using surface plasmon resonance (SPR). 3-OST-1 binds with micomolar affinity to HS (K_D = 2.79 μM), and this interaction is apparently independent of the presence of the coenzyme, 3‘-phosphoadenosine 5‘-phosphosulfate (PAPS). A conformational change in the complex has also been detected, supporting data from previous studies. Selected 3-OST-1 mutants have provided valuable information of amino acid residues that participate in 3-OST-1 interaction with HS substrate and its catalytic activity. The results from this study contribute to understanding the substrate specificity among the 3-OST isoforms and in the mechanism of 3-OST-1-catalyzed biosynthesis of anticoagulant HS.