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Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes

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dc.contributor.author Karnawat, Vishakha
dc.contributor.author Mehrotra, Sonali
dc.contributor.author Balaram, Hemalatha
dc.contributor.author Puranik, Mrinalini
dc.date.accessioned 2017-01-24T06:31:32Z
dc.date.available 2017-01-24T06:31:32Z
dc.date.issued 2016
dc.identifier.citation Karnawat, V.; Mehrotra, S.; Balaram, H.; Puranik, M., Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes. Biochemistry 2016, 55 (17), 2491-2499 http://dx.doi.org/10.1021/acs.biochem.5b01386 en_US
dc.identifier.citation Biochemistry en_US
dc.identifier.citation 55 en_US
dc.identifier.citation 17 en_US
dc.identifier.issn 0006-2960
dc.identifier.uri https://libjncir.jncasr.ac.in/xmlui/10572/2176
dc.description Restricted Access en_US
dc.description.abstract In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active Site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP.ADSS complex was found to be stable upon binding of the third ligand, hadacidin. (HDA), an analogue of L-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released froth ADSS, is unstable in solution, and converts back into, IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis. en_US
dc.description.uri http://dx.doi.org/10.1021/acs.biochem.5b01386 en_US
dc.language.iso English en_US
dc.publisher American Chemical Society en_US
dc.rights @American Chemical Society, 2016 en_US
dc.subject Biochemistry & Molecular Biology en_US
dc.subject Hypoxanthine-Guanine Phosphoribosyltransferase en_US
dc.subject Resonance Raman-Spectroscopy en_US
dc.subject Refined Crystal-Structures en_US
dc.subject Transition-State Analog en_US
dc.subject Escherichia-Coli en_US
dc.subject Ligand-Binding en_US
dc.subject Protein en_US
dc.subject Imp en_US
dc.subject Purification en_US
dc.subject Inhibition en_US
dc.title Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes en_US
dc.type Article en_US


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