CARBOXYL-TERMINUS OF INDUCIBLE NITRIC-OXIDE SYNTHASE - CONTRIBUTION TO NADPH BINDING AND ENZYMATIC-ACTIVITY

Cloning of a nitric oxide synthase (NOS) fi om RAW 264.7 mouse macroph ages (Xie, Q.-w., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225-228) yielded two sets of cDNA: one with a longer coding regio n of 1144 amino acids, whose sequence matched that of the purified pro tein, and another with a shorter coding region of 1122 amino acids, in which the last 10 carboxyl-terminal amino acids differed completely f rom those of the long form. me have now found that the short form lack s NOS activity. To determine the basis of this defect, we prepared rec ombinant chimeric, deletional, and point mutants of the long and short NOS variants, monitored their expression by immunoblot, and tested th eir enzymatic activity. By itself, lack of the 22-carboxyl-terminal re sidues of the long form NOS was scarcely consequential. Mutation of Ph e(1122), the only aromatic residue within one of the longest conserved regions shared by all NOSs of reported sequence, reduced enzymatic ac tivity by 41%. Deletion of 23 carboxyl-terminal amino acids (including Phe(1122)) reduced activity by 71%. Further loss of Ile(1121), anothe r completely conserved residue, reduced activity by 95%, and with the deletion of the rest of the conserved region, NOS activity was undetec table. Normal dimerization and binding of heme and calmodulin by the s hort variants militated against distortions of tertiary structure affe cting the amino-terminal half or middle portion of the protein. In con trast, the short variants were deficient in binding to NADPH, as predi cted by a model of tertiary structure based on that of spinach ferredo xin-NADP(+) reductase. This is the first demonstration that the carbox yl terminus of NOS is a functionally critical region.