부산대학교 도서관

Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (K[sub D]) for Atm1-C binding of ATP (K[sub D] ∼97 µM, pH 7.3, and ∼102 µM, pH 10.0) and ADP (K[sub D] ∼43 µM, pH 7.3, and 92 µM, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC[sub 50] of 10 mM. The MichaelisMenten constants V[sub max], K[sub M], and k[sub cat] of Atm1-C were measured as 1.822 µM min[sup -1], 513 µM, and 0.055 min[sup -1], respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn[sup 2+] > Mg[sup 2+] > Co[sup 2+], while Mg[sup 2+] and Co[sup 2+] were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (n[sub app] = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter. [ABSTRACT FROM AUTHOR]