Struktura, aktivita a metabolismus lidské glutamátkarboxypeptidasy II

• Main Author: Mlčochová, Petra
• Other Authors: Konvalinka, Jan
• Format: Doctoral Thesis
• Language: English
• Published: 2008
• Online Access: http://www.nusl.cz/ntk/nusl-372398

CONCLUSIONS AND PERSPECTIVES Recentry reported crystal sructures of GCpII provide stucturar insight into the organization of the substrate binding cavity and highlight residues implicated in substrate / inhibitor binding in the sI site of the enzyme. To comprement and extend the s[ucturar studies, we constructed a QMzMM model of GCPII in complex with its substrate, N-aceýt. aspartyl-glutamate, which enabled us to pÍedict additional anrino acid residues interacting with the bound subsrate. and used site-directed mutagenesis to assess the contribution of individual residues for substrate ,/ inhibitor binding and enzymatic activity of GCpII. we prepared and characterized 12 GcpJ' mutants targeting the amino acids in the vicinity of substrate./inhibitor binding pockets. The experimental results suggest that residues (especiaily Arg210) in the sť site are critical for substrate./inhibitor binding, whereas the residues forming the sl pocket niight be more important for the .fine-tuning, of GCptr substrate specificity and appear to be relevant for substrate turnover and may play a role in the enzyme's mechanism of action. Even though the QIVýMM calculations of the NAAG binding mode in the GCPII active site enabled us to predict the structure and enzyme-substrate interactions in the sl binding site, the complete...