Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli

被引:141
作者
Liu, YJ [1 ]
Thoden, JB [1 ]
Kim, J [1 ]
Berger, E [1 ]
Gulick, AM [1 ]
Ruzicka, FJ [1 ]
Holden, HM [1 ]
Frey, PA [1 ]
机构
[1] UNIV WISCONSIN,COLL AGR & LIFE SCI,DEPT BIOCHEM,MADISON,WI 53705
关键词
D O I
10.1021/bi970430a
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
Synthesis and overexpression of a gene encoding Escherichia coli UDP-galactose 4-epimerase and engineered to facilitate cassette mutagenesis are described. General acid-base catalysis at the active site of this epimerase has been studied by kinetic and spectroscopic analysis of the wild-type enzyme and its specifically mutated forms Y149F, S124A, S124V, and S124T. The X-ray crystal structure of Y149F as its abortive complex with UDP-glucose is structurally similar to that of the corresponding wild-type complex, except for the absence of the phenolic oxygen of Tyr 149. The major effects of mutations are expressed in the values of k(cat) and k(cat)/K-m. The least active mutant is Y149F, for which the value of k(cat) is 0.010% of that of the wild-type epimerase. The activity of S124A is also very low, with a k(cat) value that is 0.035% of that of the native enzyme. The values of K-m for Y149F and S124A are 12 and 21% of that of the wild-type enzyme, respectively. The value of k(cat) for S124T is about 30% of that of the wild-type enzyme, and the value of K-m is similar to that of the native enzyme. The reactivities of the mutants in UMP-dependent reductive inactivation by glucose are similarly affected, with k(obs) being decreased by 6560-, 370-, and 3.4-fold for Y149F, S124A, and S124T, respectively. The second-order rate constants for reductive inactivation by NaBH3CN, which does not require general base catalysis, are similar to that for the native enzyme in the cases of S124A, S124T, and S124V. However, Y149F reacts with NaBH3CN 12-20-fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively, The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD(+), which is present with Tyr 149 in the wild-type enzyme. The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent. The pH dependencies of A(320) showed that the pK(a) values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T. The low pK(a) value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD(+) and Lys 153 (4.5 kcal mol(-1)) and partly to hydrogen bonding with Ser 124 (1 kcal mol(-1)). The pK(a) of Tyr 149 is the same as the kinetic pK(a) for the Bronsted base that facilitates hydride transfer to NAD(+) We concluded that Tyr 149 provides the driving force for general acid-base catalysis, with Ser 124 playing an important role in mediating proton transfer.
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页码:10675 / 10684
页数:10
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共 46 条
[1]  
[Anonymous], MOL CLONING LAB MANU
[2]  
ARABSHAHI A, 1988, J BIOL CHEM, V263, P2638
[3]   Crystal structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase complexed with 17 beta-estradiol [J].
Azzi, A ;
Rehse, PH ;
Zhu, DW ;
Campbell, RL ;
Labrie, F ;
Lin, SX .
NATURE STRUCTURAL BIOLOGY, 1996, 3 (08) :665-668
[4]   EXPANSION OF THE MAMMALIAN 3-BETA-HYDROXYSTEROID DEHYDROGENASE PLANT DIHYDROFLAVONOL REDUCTASE SUPERFAMILY TO INCLUDE A BACTERIAL CHOLESTEROL DEHYDROGENASE, A BACTERIAL UDP-GALACTOSE-4-EPIMERASE, AND OPEN READING FRAMES IN VACCINIA VIRUS AND FISH LYMPHOCYSTIS DISEASE VIRUS [J].
BAKER, ME ;
BLASCO, R .
FEBS LETTERS, 1992, 301 (01) :89-93
[5]   THE IMPORTANCE OF BINDING-ENERGY IN CATALYSIS OF HYDRIDE TRANSFER BY UDP-GALACTOSE 4-EPIMERASE - A C-13 AND N-15 NMR AND KINETIC-STUDY [J].
BURKE, JR ;
FREY, PA .
BIOCHEMISTRY, 1993, 32 (48) :13220-13230
[6]   A METHOD FOR THE LIGATION OF DNA FOLLOWING ISOLATION FROM LOW MELTING TEMPERATURE AGAROSE [J].
BURNS, DM ;
BEACHAM, IR .
ANALYTICAL BIOCHEMISTRY, 1983, 135 (01) :48-51
[7]   UMP-DEPENDENT REDUCTION OF UDP-GALACTOSE 4-EPIMERASE-NAD+ COMPLEX BY SODIUM CYANOBOROHYDRIDE [J].
DAVIS, JE ;
NOLAN, LD ;
FREY, PA .
BIOCHIMICA ET BIOPHYSICA ACTA, 1974, 334 (02) :442-447
[8]   Site-directed mutagenesis of the conserved serine 138 of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme [J].
Ensor, CM ;
Tai, HH .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 220 (02) :330-333
[9]   BACTERIAL EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF 2 CRITICAL RESIDUES (TYROSINE-151 AND LYSINE-155) OF HUMAN PLACENTAL NAD(+)-DEPENDENT 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE [J].
ENSOR, CM ;
TAI, HH .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1994, 1208 (01) :151-156
[10]   REACTION OF URIDINE-DIPHOSPHATE GALACTOSE 4-EPIMERASE WITH A SUICIDE INACTIVATOR [J].
FLENTKE, GR ;
FREY, PA .
BIOCHEMISTRY, 1990, 29 (09) :2430-2436