Biochemical Characterization of Two Clinically-Relevant Human Fumarase Variants Defective for Oligomerization
Artemisa Bulku1, #, Todd M. Weaver2, Melanie B. Berkmen1, *
Identifiers and Pagination:Year: 2018
First Page: 1
Last Page: 15
Publisher Id: TOBIOCJ-12-1
Article History:Received Date: 04/10/2017
Revision Received Date: 13/12/2017
Acceptance Date: 28/12/2017
Electronic publication date: 29/01/2018
Collection year: 2018
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Fumarase, a significant enzyme of energy metabolism, catalyzes the reversible hydration of fumarate to L-malate. Mutations in the FH gene, encoding human fumarase, are associated with fumarate hydratase deficiency (FHD) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Fumarase assembles into a homotetramer, with four active sites. Interestingly, residues from three of the four subunits within the homotetramer comprise each active site. Hence, any mutation affecting oligomerization is predicted to disrupt enzyme activity.
We constructed two variants of hexahistidine-tagged human recombinant fumarase, A308T and H318Y, associated with FHD and HLRCC, respectively. Both Ala308 and His318 lie within the fumarase intersubunit interface. We purified unmodified human fumarase and the two variants, and analyzed their enzymatic activities and oligomerization states in vitro.
Both variants showed severely diminished fumarase activity. Steady-state kinetic analysis demonstrated that the variants were largely defective due to decreased turnover rate, while displaying Km values for L-malate similar to unmodified human recombinant fumarase. Blue native polyacrylamide gel electrophoresis and gel filtration experiments revealed that each variant had an altered oligomerization state, largely forming homodimers rather than homotetramers.
We conclude that A308T and H318Y render human fumarase enzymatically inactive via defective oligomerization. Therefore, some forms of FHD and HLRCC can be linked to improperly folded quaternary structure.