BINDING OF PHENOL AND ANALOGS TO ALANINE COMPLEXES OF TYROSINE PHENOL-LYASE FROM CITROBACTER-FREUNDII - IMPLICATIONS FOR THE MECHANISMS OF ALPHA,BETA-ELIMINATION AND ALANINE RACEMIZATION

被引:35
作者
CHEN, HY
PHILLIPS, RS
机构
[1] UNIV GEORGIA,DEPT CHEM,ATHENS,GA 30602
[2] UNIV GEORGIA,DEPT BIOCHEM,ATHENS,GA 30602
[3] UNIV GEORGIA,CTR MET ENZYME STUDIES,ATHENS,GA 30602
关键词
D O I
10.1021/bi00094a016
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have examined the interaction of Citrobacter freundii tyrosine phenol-lyase with both L- and D-alanine. This enzyme catalyzes the racemization of alanine as a side reaction, in addition to the physiological beta-elimination Of L-tyrosine to give phenol and ammonium pyruvate. The steady-state kinetic parameters for alanine racemization, k(cat) and K(m), for D-alanine are 0.008 s-1 and 32 mM, respectively, while those for L-alanine are 0.03 s-1 and 11 mM. Incubation of tyrosine phenol-lyase with either L- or D-alanine forms a quinonoid complex that exhibits a strong peak at 500 nm. The presence of K+ increases the intensity of the 500-nm absorption with L-alanine, but decreases the intensity of the peak with D-alanine. Rate constants for the formation of these quinonoid intermediates and the effects of phenol and analogues on the reaction with either L- or D-alanine have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Phenol binds to all the intermediates of tyrosine phenol-lyase with L- and D-alanine, but most strongly to the external aldimine complex, resulting in a decrease in the absorbance at 500 nm at equilibrium. Pyridine N-oxide binds selectively to the quinonoid complex of alanine, and thus causes an increase in the absorbance at 500 nm at equilibrium. 4-Hydroxypyridine causes a decrease in absorbance at 500 nm during the fast phase, but an increase in absorbance at 502 nm in a subsequent slow relaxation. From these studies of the effects of phenol and analogues on the reaction of tyrosine phenol-lyase with alanine, we have found there is a common quinonoid intermediate in the reactions of both L-alanine. and D-alanine. The formation of this intermediate is the rate-determining step in the racemization reaction, as the rate constants for its formation (0.01 s-1 from D-alanine and 0.034 s-1 from L-alanine) are in good agreement with the steady-state k(cat) values. The implications of these studies for the mechanisms of the beta-elimination and alanine racemization reactions are discussed.
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页码:11591 / 11599
页数:9
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