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Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits

[Image: see text] The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determin...

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Autores principales: Brandão, Tiago A. S., Richard, John P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2020
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476526/
https://www.ncbi.nlm.nih.gov/pubmed/32374983
http://dx.doi.org/10.1021/acs.biochem.0c00241
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author Brandão, Tiago A. S.
Richard, John P.
author_facet Brandão, Tiago A. S.
Richard, John P.
author_sort Brandão, Tiago A. S.
collection PubMed
description [Image: see text] The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) k(cat) and k(cat)/K(m) values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol(–1) increases in ΔG(†) for k(cat)/K(m) for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol(–1) increases, respectively, in ΔG(†). The D37G and T100′A substitutions both resulted in smaller 2 kcal mol(–1) decreases in ΔG(†) for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol(–1) compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.
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spelling pubmed-74765262021-05-06 Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits Brandão, Tiago A. S. Richard, John P. Biochemistry [Image: see text] The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) k(cat) and k(cat)/K(m) values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol(–1) increases in ΔG(†) for k(cat)/K(m) for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol(–1) increases, respectively, in ΔG(†). The D37G and T100′A substitutions both resulted in smaller 2 kcal mol(–1) decreases in ΔG(†) for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol(–1) compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change. American Chemical Society 2020-05-06 2020-06-02 /pmc/articles/PMC7476526/ /pubmed/32374983 http://dx.doi.org/10.1021/acs.biochem.0c00241 Text en Copyright © 2020 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Brandão, Tiago A. S.
Richard, John P.
Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title_full Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title_fullStr Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title_full_unstemmed Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title_short Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
title_sort orotidine 5′-monophosphate decarboxylase: the operation of active site chains within and across protein subunits
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476526/
https://www.ncbi.nlm.nih.gov/pubmed/32374983
http://dx.doi.org/10.1021/acs.biochem.0c00241
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