Mechanism of Inactivation of Inducible Nitric Oxide Synthase by Amidines. Irreversible Enzyme Inactivation without Inactivator Modification

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• 作者：Yaoqiu Zhu ; Dejan Nikolic ; Richard B. Van Breemen ; and Richard B. Silverman
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：January 26, 2005
• 年：2005
• 卷：127
• 期：3
• 页码：858 - 868
• 全文大小：238K
• 年卷期：v.127,no.3(January 26, 2005)
• ISSN：1520-5126

文摘

Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrullineand nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding,and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400Wwas recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected.Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator ofiNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W showsboth loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation byanother amidine-containing compound, N⁵-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdinproduced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation ofiNOS with either 1400W or L-NIO to be biliverdin IX out of the four possible regioisomers. Two mechanismswere previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate,leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methylgroup followed by attachment to the heme cofactor, which causes the enzyme to catalyze the hemeoxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d₃-1400W,which produced no d₂-1400W. Detection of carbon monoxide as one of the heme-degradation productsfurther excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanismis proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because ofthe amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing itsconversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one thatresembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IX. This appears tobe the first example of a compound that causes irreversible inactivation of an enzyme without itself becomingmodified in any way.