MODULAR STRUCTURE OF NEURONAL NITRIC-OXIDE SYNTHASE - LOCALIZATION OFTHE ARGININE BINDING-SITE AND MODULATION BY PTERIN

A putative dihydrofolate reductase (DHFR) module has been identified i n neuronal nitric oxide synthase, consisting of amino acids 558-721, a nd is proposed to be the site of tetrahydrobiopterin (BH4) binding. Th is polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The prote in binds N-omega-nitro-L-arginine (NNA) tightly, but this binding is n ot stimulated by BH4. cDNAs for Module II (residues 220-557) and Modul e III (residues 220-721) have been expressed as fusion proteins with G ST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are ta ken as strong evidence that the DHFR module contains the L-arginine bi nding site and, presumably, the BH4 binding site by analogy to its hom ology with DHFR, but that tight binding of BH4 requires amino acids 22 0-577. (C) 1995 Academic Press, Inc.