Characterization of the Putative GTP-Binding Site Residues of Escherichia coli Adenylosuccinate Synthetase by Site-Directed Mutagenesis

Abstract

Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins. This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase. On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21 ras proteins are different. Yet, residues that interact with GTP in the p21 ras proteins are present in the synthetase in nearly identical positions. We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and R331L and R331R, two mutants of Lys 331 that are involved in guanine ring binding. The K m values for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in the K m values for IMP. Because both mutations affected the K m values for GTP similarly, whereas the k cat and secondary structure were essentially unchanged, it is suggested that Lys 331 is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N-xi of the Lys is not necessarily important in GTP-binding on the enzyme. Therefore, Lys 331 may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry. The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates. The NMR spectra of both enzymes were also similar in the absence of substrates. The dissociation constant ( K d ) for IMP of the G15V mutant was 4.8-fold larger than its K m value which was 1.5-fold increased compared to the wildtype enzyme. From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and LMP binding depend on the presence of the other substrate at the active site of the enzyme. These results suggest that the Lys 331 of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins. However, the phosphate-binding loop has additional functions besides the interaction with the gamma-phosphate of GTP.