We recently reported that glutamate carboxypeptidase II (GCPII) has a new physiological function degrading amyloid-beta (A beta), distinct from its own hydrolysis activity in N-acetyl-L-aspartyl-L-glutamate (NAAG); however, its underlying mechanism remains undiscovered. Using site-directed mutagenesis and S1 pocket-specific chemical inhibitor (compound 2), which was developed for the present study based on in silico computational modeling, we discovered that the A beta degradation occurs through S1 pocket but not through S1' pocket responsible for NAAG hydrolysis. Treatment with compound 2 prevented GCPII from A beta degradation without any impairment in NAAG hydrolysis. Likewise, 2-PMPA (specific GCPII inhibitor developed targeting S1' pocket) completely blocked the NAAG hydrolysis without any effect on A beta degradation. Pre-incubation with NAAG and A beta did not affect A beta degradation and NAAG hydrolysis, respectively. These data suggest that GCPII has two distinctive binding sites for two different substrates and that A beta degradation occurs through binding to S1 pocket of GCPII. (C) 2013 Published by Elsevier Inc.