Enzyme I of the phosphotransferase system: induced-fit protonation of the reaction transition state by Cys-502
| Autor: | Bernhard Erni, Luis F. García-Alles, Ignacio Alfonso |
|---|---|
| Přispěvatelé: | Universidad de Oviedo, Universidad de Oviedo [Oviedo] |
| Rok vydání: | 2003 |
| Předmět: |
[SDV.BIO]Life Sciences [q-bio]/Biotechnology
Protein Conformation Biochemistry Phosphoenolpyruvate chemistry.chemical_compound MESH: Protein Conformation MESH: Structure-Activity Relationship Enzyme Inhibitors 0303 health sciences Oxalates MESH: Kinetics Chemistry 030302 biochemistry & molecular biology PEP group translocation MESH: Oxalates MESH: Enzyme Inhibitors MESH: Phosphoenolpyruvate cardiovascular system Protons Phosphoenolpyruvate carboxykinase Dimerization Steric effects MESH: Mutation Stereochemistry education Substituent Protonation MESH: Phosphoenolpyruvate Sugar Phosphotransferase System Oxalate Catalysis 03 medical and health sciences Structure-Activity Relationship Isomerism MESH: Isomerism [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Cysteine Binding site Phosphoenolpyruvate Sugar Phosphotransferase System 030304 developmental biology Binding Sites MESH: Phosphotransferases (Nitrogenous Group Acceptor) Phosphotransferases (Nitrogenous Group Acceptor) Substrate (chemistry) MESH: Cysteine MESH: Catalysis Kinetics MESH: Binding Sites MESH: Dimerization Mutation MESH: Protons |
| Zdroj: | Biochemistry Biochemistry, American Chemical Society, 2003, 42 (16), pp.4744-4750. ⟨10.1021/bi034007f⟩ |
| ISSN: | 0006-2960 1520-4995 |
| Popis: | International audience; Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal domain with the phosphorylation site (His-189) and a C-terminal domain with the PEP binding site. Here we use C3-substituted PEP analogues as substrates and inhibitors and the EI(C502A) mutant to characterize structure-activity relationships of the PEP binding site. EI(C502A) is 10 000 times less active than wild-type EI [EI(wt)] with PEP as the substrate, whereas the two forms are equally active with ZClPEP. Cys-502 acts as an acid-base catalyst which stereospecifically protonates the pyruvoyl enolate at C3. The electron-withdrawing chlorine of ZClPEP can compensate for the lack of Cys-502, and in this case, the released 3-Cl-enolate is protonated nonstereospecifically. Several PEP analogues were assayed as inhibitors and as substrates. The respective K(I)/K(m) ratios vary between 3 and 40 for EI(wt), but they are constant and around unity for EI(C502A). EI(wt) with PEP as the substrate is inhibited by oxalate, whereas EI(C502A) with ZClPEP is not. The different behavior of EI(wt) and EI(C502A) toward the PEP analogues and oxalate suggests that the PEP binding site of EI(wt) exists in a "closed" and an "open" form. The open to closed transition is triggered by the interaction of the substrate with Cys-502. The closed conformation is sterically disfavored by C3-modified substrate analogues such as ZClPEP and ZMePEP. If site closure does not occur as with EI(C502A) and bulky substrates, the transition state is stabilized by electron dispersion to the electron-withdrawing substituent at C3. |
| Databáze: | OpenAIRE |
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