Structural and Biochemical Characterization of the Folyl-poly-γ-L-glutamate Hydrolysing Activity of Human Glutamate Carboxypeptidase II

Michal Navratil¹, Jakub Ptacek², Jana Starkova¹, Cyril Barinka², Jacek Lubkowski³, Jan Konvalinka^1,4

¹Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic, 16610

²Institute of Biotechnology, Prague, Czech Republic, 14220

³Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA

⁴Department of Biochemistry, Charles University, Prague, Czech Republic, 12800

Human glutamate carboxypeptidase II (GCPII) acts as folate hydrolase in proximal small intestine, participating thus in the absorption of dietary folates by humans. Additionally, there are conflicting genotype-phenotype association data linking the GCPII H475Y variant to changed blood-levels of folate or homocysteine and to various pathologies. Despite the importance of GCPII as a folate hydrolase there is limited the knowledge of folate recognition and processing by the wild-type enzyme as well as virtually no information on the structural and functional consequence of GCPII H475Y polymorphism on folate hydrolysis. Here we present a series of detailed structural, kinetic and mutagenesis studies aimed at dissecting GCPII role in folate metabolism.

We first determined the X-ray structure of the GCPII H475Y variant at 1.70 Å resolution and using a newly introduced UPLC assay we compared the H475Y folate hydrolyzing activity on a panel of folyl-poly-γ-L-glutamates to the wild-type enzyme. Combined structural and kinetic data suggest near identity of both GCPII variants, putting into question a direct link between GCPII enzymatic activity and physiological levels of folate metabolites. To gain a deeper insight how GCPII recognizes and interacts with different folate species, we also determined X-ray structures of complexes between the inactive GCPII(E424A) mutant and a panel of naturally occurring polyglutamylated folates. The structural data revealed details of GCPII-folate interactions including the surprising engagement of the previously identified “arene-binding site” by the pteroate group of folates. To support and extend our structural observations, mutants of the GCPII “arene-binding site” W541A, R511L and R463L were produced and characterized in terms of the hydrolysis of the folyl-poly-γ-L-glutamates and inhibition profiles of pteroic acid.