Active Site Rearrangement of the 2-Hydrazinopyridine Adduct in Escherichia coli Amine Oxidase to an Azo Copper(II) Chelate Form: A Key Role for Tyrosine 369 in Controlling the Mobility of the T

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• 作者：Minae Mure ; Christian R. Kurtis ; Doreen E. Brown ; Melanie S. Rogers ; Winston S. Tambyrajah ; Colin Saysell ; Carrie M. Wilmot ; Simon E. V. Phillips ; Peter F. Knowles ; David M. Dooley ; and Michael J. McP
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：February 8, 2005
• 年：2005
• 卷：44
• 期：5
• 页码：1583 - 1594
• 全文大小：468K
• 年卷期：v.44,no.5(February 8, 2005)
• ISSN：1520-4995

文摘

Adduct I (es/gifchars/lambda.gif" BORDER=0 >_max at ~430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and theTPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH,25 es/entities/deg.gif">C, but slowly converts to another spectroscopically distinct species with a es/gifchars/lambda.gif" BORDER=0 >_max at ~530 nm (adductII) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 es/entities/deg.gif">C or byincreasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectralchanges similar to WT when incubated at 60 es/entities/deg.gif">C. By contrast, in the Y369F mutant adduct I was notstable at pH 7, 25 es/entities/deg.gif">C, and gradually converted to adduct II, and this rate of conversion was faster at pH9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on theUV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly,it was found that addition of Cu²⁺ to 2 at pH 7 gave a product (3) that exhibited almost identicalspectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinatedform of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results ledto the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu²⁺.The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPRspectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearlydemonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369)is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimalorientation for reactivity with substrate amines.