Solution structure of ligands involved in purine salvage pathway

Karnawat, Vishakha; Puranik, Mrinalini

doi:10.1016/j.saa.2015.07.013

Cited by 1 publication

(4 citation statements)

References 31 publications

(37 reference statements)

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“…The resultant spectrum, containing bands only from the new species, was compared with the Raman spectrum of 6-pIMP computed using density functional theory. The computed Raman wavenumbers are in excellent agreement with experimental values . We further compared the UVRR spectra of AMP with that of 6-pIMP (Figure S2).…”

Section: Resultssupporting

confidence: 71%

“…UVRR spectra of enzyme·nucleotide complexes were acquired at a Raman excitation wavelength of 260 nm (Figure ). Vibrational modes of nucleobases are resonance-enhanced at this excitation wavelength, while spectra of the protein, sugar, and phosphate do not contribute significantly to the Raman signal because of their small cross section at this wavelength. − In previous work, we have demonstrated that resonance Raman spectra of enzyme-bound nucleotides and nucleobases report on the protonation state and noncovalent interactions of the ligand. , In this work, we report on perturbations in the structure of bound ligands at the active site of MjADSS through observed spectral shifts and intensity modulation in the UVRR spectra. We obtained UVRR spectra of bound ligands by subtracting the spectra of the apoenzyme and unbound ligands from those of the enzyme–ligand complexes (see Data Analysis).…”

Section: Resultsmentioning

confidence: 79%

“…27−29 In previous work, we have demonstrated that resonance Raman spectra of enzyme-bound nucleotides and nucleobases report on the protonation state and noncovalent interactions of the ligand. 2,29 In this work, we report on perturbations in the structure of bound ligands at the active site of MjADSS through observed spectral shifts and intensity modulation in the UVRR spectra. We obtained UVRR spectra of bound ligands by subtracting the spectra of the apoenzyme and unbound ligands from those of the enzyme−ligand complexes (see Data Analysis).…”

Section: ■ Resultsmentioning

confidence: 99%

“…The computed Raman wavenumbers are in excellent agreement with experimental values. 29 We further compared the UVRR spectra of AMP with that of 6-pIMP (Figure S2). AMP serves as a stable model for 6-pIMP as a purine with the sp 3 carbon at C6 because of the similarity in their structures that arises from, first, the presence of a single bond between C6 and the exocyclic group attached to it and, second, the absence of any hydrogen at the N1 position that results in similar purine ring properties in 6-pIMP, AMP, and deprotonated IMP.…”

Section: ■ Resultsmentioning

confidence: 99%

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

et al. 2016

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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 from 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.

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Section: Resultssupporting

confidence: 71%