Please use this identifier to cite or link to this item: http://lib.jncasr.ac.in:8080/jspui/handle/10572/2176
Title: Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes
Authors: Karnawat, Vishakha
Mehrotra, Sonali
Balaram, Hemalatha
Puranik, Mrinalini
Keywords: Biochemistry & Molecular Biology
Hypoxanthine-Guanine Phosphoribosyltransferase
Resonance Raman-Spectroscopy
Refined Crystal-Structures
Transition-State Analog
Escherichia-Coli
Ligand-Binding
Protein
Imp
Purification
Inhibition
Issue Date: 2016
Publisher: American Chemical Society
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
Biochemistry
55
17
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.
Description: Restricted Access
URI: http://hdl.handle.net/10572/2176
ISSN: 0006-2960
Appears in Collections:Research Papers (Hemalatha Balaram)

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