Skip to content

Research at St Andrews

Mechanism of the human nucleocytoplasmic hexosaminidase D

Research output: Contribution to journalArticle


Matthew G Alteen, Verena Oehler, Ivana Nemčovičová, Iain B H Wilson, David J Vacadlo, Tracey Maureen Gloster

School/Research organisations


Mammalian β-hexosaminidases have emerged as playing essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these β-hexosaminidases, Hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized β-hexosaminidase and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild type and mutant enzymes, linear free energy analyses of the enzyme catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies, collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second order rate constant at pH 6.5-7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki of 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to better understand the physiological role played by this unusual enzyme.


Original languageEnglish
Pages (from-to)2753-2747
Issue number19
Early online date5 May 2016
StatePublished - 17 May 2016

    Research areas

  • Hexosaminidase, Glycoside hydrolase, Enzyme kinetics, Enzyme mechanism, Enzyme inhibition, Substrate-assisted catalysis

Discover related content
Find related publications, people, projects and more using interactive charts.

View graph of relations

Related by author

  1. New irreversible α-L-iduronidase inhibitors and activity-based probes

    Artola, M., Kuo, C-L., McMahon, S. A., Oehler, V., Hansen, T., van der Lienden, M., He, X., van den Elst, H., Florea, B. I., Kermode, A. R., Gloster, T. M., van der Marel, G. A., Codée, J. D. C., Overkleeft, H. S. & Aerts, J. M. F. G. 11 Oct 2018 (Accepted/In press) In : Chemistry - A European Journal. In press

    Research output: Contribution to journalArticle

  2. Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level

    Ren, W., Pengelly, R. J., Farren-Dai, M., Abadi, S. S. K., Oehler, V., Akintola, O., Draper, J., Meanwell, M., Chakladar, S., Swiderek, K., Moliner, V., Britton, R., Gloster, T. & Bennet, A. 13 Aug 2018 In : Nature Communications. 9, 12 p., 3243

    Research output: Contribution to journalArticle

  3. Metabolic inhibitors of O-GlcNAc transferase (OGT) that act in vivo implicate decreased O-GlcNAc levels in leptin-mediated nutrient sensing

    Liu, T., Zandberg, W. F., Gloster, T. M., Deng, L., Murray, K. D., Shan, X. & Vocadlo, D. J. 25 Jun 2018 In : Angewandte Chemie International Edition. 57, 26, p. 7644-7648

    Research output: Contribution to journalArticle

  4. The enzymatic degradation of heparan sulfate

    Griffin, L. S. & Gloster, T. M. 1 Aug 2017 In : Protein & Peptide Letters. 24, 8, p. 710-722

    Research output: Contribution to journalReview article

  5. Structural snapshots for mechanism-based inactivation of a glycoside hydrolase by cyclopropyl-carbasugars

    Adamson, C., Pengelly, R. J., Kazem Abadi, S. S., Chakladar, S., Draper, J., Britton, R., Gloster, T. M. & Bennet, A. J. 21 Nov 2016 In : Angewandte Chemie International Edition. 55, 48, p. 14978-14982

    Research output: Contribution to journalArticle

Related by journal

  1. Allosteric activation shifts the rate-limiting step in a short-form ATP phosphoribosyltransferase

    Fisher, G., Thomson, C. M., Stroek, R., Czekster, C. M., Hirschi, J. S. & da Silva, R. G. 24 Jul 2018 In : Biochemistry. 57, 29, p. 4357-4367

    Research output: Contribution to journalArticle

  2. Oxidation of the cyanobactin precursor peptide is independent of the leader peptide and operates in a defined order

    Gao, S., Ge, Y., Bent, A. F., Schwarz-Linek, U. & Naismith, J. H. 16 Oct 2018 In : Biochemistry. 57, 41, p. 5996-6002 7 p.

    Research output: Contribution to journalArticle

  3. Adenosine monophosphate binding stabilizes the KTN domain of the Shewanella denitrificans Kef potassium efflux system

    Pliotas, C., Grayer, S. C., Ekkerman, S., Chan, A. K. N., Healy, J., Marius, P., Bartlett, W., Khan, A., Cortopassi, W. A., Chandler, S. A., Rasmussen, T., Benesch, J. L. P., Paton, R. S., Claridge, T. D. W., Miller, S., Booth, I. R., Naismith, J. H. & Conway, S. J. 15 Aug 2017 In : Biochemistry. 56, 32, p. 4219-4234

    Research output: Contribution to journalArticle

  4. Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

    Czekster, C. M. & Naismith, J. H. 18 Apr 2017 In : Biochemistry. 56, 15, p. 2086-2095 10 p.

    Research output: Contribution to journalArticle

  5. Kinetics and structure of a cold-adapted hetero-octameric ATP phosphoribosyltransferase

    Stroek, R., Ge, Y., Talbot, P. D., Glok, M. K., Bernas, K. E., Thomson, C. M., Gould, E. R., Alphey, M. S., Liu, H., Florence, G. J., Naismith, J. H. & da Silva, R. G. 7 Feb 2017 In : Biochemistry. 56, 5, p. 793-803 11 p.

    Research output: Contribution to journalArticle

ID: 242458071