Identification of the substrate binding sites within the yeast mitochondrial citrate transport protein.
The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K(m) values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K(m) values (i.e. 5 to >300-fold). Each of these mutants displayed V(max) values that were reduced by > or = 98% and resultant catalytic efficiencies that were reduced by > or = 99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.
Ma
Chunlong
C
Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois 60064, USA.
Remani
Sreevidya
S
Sun
Jiakang
J
Kotaria
Rusudan
R
Mayor
June A
JA
Walters
D Eric
DE
Kaplan
Ronald S
RS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D002352:Carrier Proteins; C020723:citrate-binding transport protein