Abstract
Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis. The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics. CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable. However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme. Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate. The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305.
Highlights
Adenylosuccinate is cleaved by adenylosuccinate lyase to form AMP and fumarate
The enzyme from E. coli was first purified to homogeneity in 1976 [2]
The Km for aspartate was obtained by holding GTP and IMP at their saturating concentrations, whereas the K m values for GTP and IMP were obtained by using an aspartate concentration of about 30 mM. 1-80 JLg of the purified enzymes were used in each kinetic assay reaction, depending on the specific activity of the enzyme
Summary
13160-13163, 1995 Printed in U.S.A. Identification of Arginine Residues in the Putative L-Aspartate Binding Site of Escherichia coli Adenylosuccinate Synthetase*. Adenylosuccinate synthetase (AMPSase)l from Escherichia coli (lMP:L-aspartate ligase (GTP-forming), EC 6.3.4.4.) catalyzes the first committed step in the de novo synthesis of AMP from IMP. Our results strongly suggest that the three arginine residues are not part of the GTP or IMP binding sites but rather are involved in aspartate binding and to some extent in catalysis as well. This is the first study that implicates specific residues in the binding of aspartate to AMPSase
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
