SITE-DIRECTED MUTAGENESIS OF HIS343-]ALA IN CITROBACTER-FREUNDII TYROSINE PHENOL-LYASE - EFFECTS ON THE KINETIC MECHANISM AND RATE-DETERMINING STEP

被引:3
作者
CHEN, HY
GOLLNICK, P
PHILLIPS, RS
机构
[1] UNIV GEORGIA,DEPT CHEM,ATHENS,GA 30602
[2] UNIV GEORGIA,DEPT BIOCHEM,ATHENS,GA 30602
[3] UNIV GEORGIA,CTR METALLOENZYME STUDIES,ATHENS,GA 30602
[4] SUNY BUFFALO,DEPT BIOL SCI,BUFFALO,NY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1995年 / 229卷 / 02期
关键词
TYROSINE PHENOL-LYASE; TRYPTOPHAN INDOLE-LYASE; L-TYROSINE; 3-FLUORO-L-TYROSINE; REACTION MECHANISM;
D O I
10.1111/j.1432-1033.1995.0540k.x
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
His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. and Wilson, K. S. (1993) Biochemistry 32, 4195-4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha,beta-elimination (except S-ethyl-L-cysteine) exhibited lower k(cat) (10 - 30 %) and k(cat)/K-m (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. and Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of k(cat)\K-m for this reaction exhibits two pK(a) values with an average of 7.7+/-0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of k(cat)/K-m for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pK(a) Values with an average of 7.8+/-0.3. However, k(cat) for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pK(a) values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on k(cat) and k(cat)/K-m for 3-fluoro-L-[alpha-H-2]-tyrosine are independent of pH and equal to 3.9+/-0.2 and 2.2+/-0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4+/-0.2 and 3.8+/-0.3, respectively. Based on these observations and the pH data, we conclude that alpha-proton abstraction is apparently the rate-determining step in the reaction of 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase; however, quinonoid intermediate formation (140 s(-1)) is much faster than k(cat) of 3-fluoro-L-tyrosine (1.4 s(-1)) for wild-type tyrosine phenol-lyase; thus, the mutation of His343-->Ala has apparently changed the rate-determining step of the reaction with 3-fluoro-L-tyrosine. L-Methionine, L-homoserine, L-phenylalanine and L-alanine form quinonoid intermediates with wild-type tyrosine phenol-lyase which exhibit absorbance peaks at about 500 nm but the intensity of these absorbance peaks is dramatically reduced for the mutant. Single-wavelength stopped-flow kinetic measurements demonstrate that [H343A]tyrosine phenol-lyase exhibits slower rate constants for alpha-deprotonation and faster rate constants for reprotonation than does the wild-type. Taken together, these results demonstrate that, while His343 is not an essential basic group in tyrosine phenol-lyase, it does play an important function in catalysis, possibly by facilitating the conformational change from an 'open' to 'closed' form when substrates bind.
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收藏
页码:540 / 549
页数:10
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