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J. Biol. Chem., Vol. 279, Issue 41, 42566-42573, October 8, 2004

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Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity^*

Christian Götting, Sandra Müller, Manuela Schöttler, Sylvia Schön, Christian Prante, Thomas Brinkmann, Joachim Kuhn, and Knut Kleesiek

From the Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrae 11, 32545 Bad Oeynhausen, Germany

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314–316 and 745–747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the ³¹⁴DED³¹⁶ motif did not have any effect on enzyme activity, whereas alterations of the ⁷⁴⁵DWD⁷⁴⁷ motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp⁷⁴⁵ to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (K_m = 6.9 µM) and D747E (K_m = 4.4 µM) in comparison with the wild-type enzyme (K_m = 0.9 µM). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower V_max, with K_m values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. K_i values for inhibition by UDP were determined to be 1.9–24.6 µM for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the K_i constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

Received for publication, February 6, 2004 , and in revised form, August 3, 2004.

^* The work was supported by Deutsche Forschungsgemeinschaft Grant BR1226/5-2 and the Stiftung für Pathobiochemie und Molekulare Diagnostik. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-5731-972-033; Fax: 49-5731-972-013; E-mail: cgoetting{at}hdz-nrw.de.

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