Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.
- MeSH
- alanin metabolismus MeSH
- biologické modely MeSH
- dipeptidy genetika metabolismus MeSH
- financování organizované MeSH
- glutamátkarboxypeptidasa II genetika chemie metabolismus MeSH
- hydrolýza MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutageneze MeSH
- substituce aminokyselin MeSH
- substrátová specifita genetika MeSH
- termodynamika MeSH
- vazba proteinů genetika MeSH
- vazebná místa genetika MeSH
- vodíková vazba MeSH
- výpočetní biologie metody MeSH
- zinek chemie MeSH
- Check Tag
- lidé MeSH
- MeSH
- dipeptidy fyziologie genetika MeSH
- fyziologie výživy fyziologie MeSH
- kyselina askorbová farmakokinetika imunologie terapeutické užití MeSH
- lidé MeSH
- peroxidace lipidů genetika účinky záření MeSH
- poškození DNA fyziologie genetika imunologie MeSH
- volné radikály krev metabolismus škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
