Title : Reaction mechanism of
glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods
Abstract :
- 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