The chondroitin polymerase K4CP and the molecular mechanism of selective bindings of donor substrates to two active sites

Mack Sobhany, Yoshimitsu Kakuta, Nobuo Sugiura, Koji Kimata, Masahiko Negishi

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Bacterial chondroitin polymerase K4CP is a multifunctional enzyme with two active sites. K4CP catalyzes alternative transfers of glucoronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to elongate a chain consisting of the repeated disaccharide sequence GlcAβ1-3GalNAcβ1-4. Unlike the polymerization reactions of DNA and RNA and polypeptide synthesis, which depend upon templates, the monosaccharide polymerization by K4CP does not. To investigate the catalytic mechanism of this reaction, we have used isothermal titration calorimetry to determine the binding of the donor substrates UDP-GlcA and UDP-GalNAc to purified K4CP protein and its mutants. Only one donor molecule bound to one molecule of K4CP at a time. UDP-GlcA bound only to the C-terminal active site at a high affinity (Kd = 6.81 μM), thus initiating the polymerization reaction. UDP-GalNAc could bind to either the N-terminal or C-terminal active sites at a low affinity (Kd = 266-283 μM) but not to both sites at the same time. The binding affinity of UDP-GalNAc to a K4CP N-terminal fragment (residues 58-357) was profoundly decreased, yielding the average Kd value of 23.77 μM, closer to the previously reported Km value for the UDP-GalNAc transfer reaction that takes place at the N-terminal active site. Thus, the first step of the reaction appears to be the binding of UDP-GlcA to the C-terminal active site, whereas the second step involves the C-terminal region of the K4CP molecule regulating the binding of UDP-GalNAc to only the N-terminal active site. Alternation of these two specific bindings advances the polymerization reaction by K4CP.

Original languageEnglish
Pages (from-to)32328-32333
Number of pages6
JournalJournal of Biological Chemistry
Volume283
Issue number47
DOIs
Publication statusPublished - Nov 21 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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