Structural Requirements for Human Inducible Nitric Oxide Synthase Substrates and Substrate Analogue Inhibitors

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• 作者：Stephan K. Grant ; Barbara G. Green ; Janet Stiffey-Wilusz ; Philippe L. Durette ; Shrenik K. Shah ; and John W. Kozarich
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：March 24, 1998
• 年：1998
• 卷：37
• 期：12
• 页码：4174 - 4180
• 全文大小：319K
• 年卷期：v.37,no.12(March 24, 1998)
• ISSN：1520-4995

文摘

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39)catalyzes the NADPH-dependentoxidation of one of the free guanidino nitrogens of L-Argto form nitric oxide and L-citrulline.Analoguesof L-Arg and the inhibitor,L-N⁶-(1-iminoethyl)lysine, wereused to define structural elements required forthe binding and catalysis of compounds. L-Arganalogues with sequentially shorter methylene spacingbetween the guanidino group and the amino acid portion of the moleculewere not iNOS substrates butwere reversible inhibitors. L-Arg analogues such asagmatine with a hydroxyl substitution at the 2-aminoposition were substrates. Desaminoarginine was not a substrate buta reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give typeI difference spectra similar tothat of L-Arg. The amidino compoundsL-N⁶-(1-iminoethyl)lysine,L-N⁵-(1-iminoethyl)ornithine,and N⁵-(1-iminoethyl)cadaverdine, but notN⁶-(1-iminoethyl)-6-aminocaproic acid, wereNADPH-dependent,irreversible inactivators of iNOS. For both the L-Argand L-N⁶-(1-iminoethyl)lysineanalogues, the 2-aminogroup appeared to play an important role in catalytic events leading toeither substrate turnover ormechanism-based inactivation. Inactivation of iNOS byL-N⁶-(1-iminoethyl)lysine wasNADPH- anddioxygen-dependent, but low incorporation of radiolabel withDL-[4,5-³H]-N⁶-(1-iminoethyl)lysineindicatesthat the mechanism of enzyme inactivation is not covalent modificationof the protein.