Conserved Tyrosine-369 in the Active Site of Escherichia coli Copper Amine Oxidase Is Not Essential
详细信息 查看全文
- 作者：Jeremy M. Murray ; Christian R. Kurtis ; Winston Tambyrajah ; Colin G. Saysell ; Carrie M. Wilmot ; Mark R. Parsons ; Simon E. V. Phillips ; Peter F. Knowles ; and Michael J. McPherson
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：October 30, 2001
- 年：2001
- 卷：40
- 期：43
- 页码：12808 - 12818
- 全文大小：204K
- 年卷期：v.40,no.43(October 30, 2001)
- ISSN：1520-4995
文摘
Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosineby a single turnover mechanism; second, the oxidative deamination of primary amine substrates with theproduction of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme. The importanceof active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms. One conserved residue is a tyrosine, Tyr369 inthe Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ. To explore theimportance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to aphenylalanine. We have determined the X-ray crystal structure of this variant enzyme to 2.1 Å resolution,which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reactionof the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivityof Y369F compared with wild type with more efficient formation of an adduct (
fchars/lambda.gif" BORDER=0 >max = 525 nm) perhapsreflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP alsoreveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is notessential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-visspectrum of the Y369F protein shows a broader peak and red-shifted fchars/lambda.gif" BORDER=0 >max at 496 nm compared with wildtype (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurementsreveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the KM for substratefchars/beta2.gif" BORDER=0 ALIGN="middle">-phenethylamine increases significantly, apparently due to an elevated pKa (5.75-6.5) for the catalyticbase, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, theKM for wild type and Y369F are similar at 1.2 and 1.5 
f">M, respectively, while kcat is decreased from 15s-1 in wild type to 0.38 s-1, resulting in a 50-fold decrease in kcat/KM for Y369F. Transient kineticsexperiments indicate that while the initial stages of enzyme reduction are slower in the variant, these donot represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as acomponent of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observedfor the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can becompensated for efficiently within the active site.
fchars/lambda.gif" BORDER=0 >max = 525 nm) perhapsreflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP alsoreveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is notessential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-visspectrum of the Y369F protein shows a broader peak and red-shifted fchars/lambda.gif" BORDER=0 >max at 496 nm compared with wildtype (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurementsreveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the KM for substratefchars/beta2.gif" BORDER=0 ALIGN="middle">-phenethylamine increases significantly, apparently due to an elevated pKa (5.75-6.5) for the catalyticbase, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, theKM for wild type and Y369F are similar at 1.2 and 1.5 f">M, respectively, while kcat is decreased from 15s-1 in wild type to 0.38 s-1, resulting in a 50-fold decrease in kcat/KM for Y369F. Transient kineticsexperiments indicate that while the initial stages of enzyme reduction are slower in the variant, these donot represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as acomponent of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observedfor the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can becompensated for efficiently within the active site.