RESUMO
Pyruvate decarboxylase (PDC) uses thiamine diphosphate as an essential cofactor to catalyze the formation of acetaldehyde on the pathway of ethanol synthesis. Here we report the crystallographic image of a prereaction intermediate of a bacterial pyruvate decarboxylase prepared by cocrystallizing the enzyme with pyruvate and a stable analogue of the cofactor's activated ylid form. A second crystal structure of PDC in complex with fluoride shows that the ion organizes a water molecule that occludes the pyruvate binding site, accounting for the inhibitory effect of the halide. Also reported is a structure of the cofactor-free apo form, which when compared to the structure of the holo form indicates how thiamine diphosphate organizes the active site pocket of pyruvate decarboxylase to support catalysis. Guided by the structural and enzymatic data, we propose roles for several key residues in the catalytic mechanism.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Piruvato Descarboxilase/química , Piruvato Descarboxilase/metabolismo , Zymomonas/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Fluoretos/química , Fluoretos/metabolismo , Estrutura Secundária de Proteína , Piruvato Descarboxilase/genética , Ácido Pirúvico/química , Ácido Pirúvico/metabolismoRESUMO
Novel triazole-based pyrophosphate analogues of thiamine pyrophosphate (TPP) have been synthesised and tested for inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis. The thiazolium ring of thiamine was replaced by a triazole in an efficient two-step procedure. Pyrophosphorylation then gave extremely potent triazole inhibitors with K(I) values down to 20 pM, compared to a K(D) value of 0.35 microM for TPP. This triazole scaffold was used for further investigation and six analogues containing mimics of the pyrophosphate group were synthesised and tested for inhibition of PDC. Several effective analogues were found with K(I) values down to around 1 nM.
Assuntos
Difosfatos/química , Piruvato Descarboxilase/antagonistas & inibidores , Tiamina Pirofosfato/síntese química , Tiamina Pirofosfato/farmacologia , Triazóis/química , Zymomonas/enzimologia , Difosfatos/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Tiamina Pirofosfato/análogos & derivados , Tiamina Pirofosfato/metabolismoRESUMO
Replacement of the thiazolium ring of thiamine pyrophosphate with a triazole gives extremely potent inhibitors of pyruvate decarboxylase from Z. mobilis, with K(I) values down to 20 pM; this system was used to explore pyrophosphate mimics and several effective analogues were discovered.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Piruvato Descarboxilase/antagonistas & inibidores , Tiamina Pirofosfato/análogos & derivados , Triazóis/síntese química , Triazóis/farmacologia , Zymomonas/enzimologia , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/farmacologia , Cinética , Piruvato Descarboxilase/metabolismo , Tiamina Pirofosfato/síntese química , Tiamina Pirofosfato/farmacologiaRESUMO
It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon-carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate-cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.