Substrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site.

TitleSubstrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site.
Publication TypeJournal Article
Year of Publication2012
AuthorsMonné, M, Miniero, DValeria, Daddabbo, L, Robinson, AJ, Kunji, ERS, Palmieri, F
JournalJ Biol Chem
Volume287
Issue11
Pagination7925-34
Date Published2012 Mar 09
ISSN1083-351X
KeywordsAmino Acid Substitution, Amino Acid Transport Systems, Basic, Binding Sites, Biological Transport, Active, Humans, Mitochondrial Proteins, Mutation, Missense, Ornithine, Protein Isoforms, Structure-Activity Relationship, Substrate Specificity
Abstract

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.

DOI10.1074/jbc.M111.324855
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.M111.324855
PubMed ID22262851
PubMed Central IDPMC3318746
Grant ListMC_U105663139 / / Medical Research Council / United Kingdom
MC_U105674181 / / Medical Research Council / United Kingdom