TY - JOUR
T1 - Modular structure of neuronal nitric oxide synthase
T2 - Localization of the arginine binding site and modulation by pterin
AU - Nishimura, Jonathan S.
AU - Martasek, Pavel
AU - McMillan, Kirk
AU - Salerno, John C.
AU - Liu, Qing
AU - Gross, Steven S.
AU - Masters, Bettie Sue Siler
PY - 1995/5/16
Y1 - 1995/5/16
N2 - A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds Nω-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.
AB - A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds Nω-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.
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U2 - 10.1006/bbrc.1995.1659
DO - 10.1006/bbrc.1995.1659
M3 - Article
C2 - 7538758
AN - SCOPUS:0029033858
VL - 210
SP - 288
EP - 294
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -