Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

被引:526
作者
Vocadlo, DJ
Davies, GJ
Laine, R
Withers, SG [1 ]
机构
[1] Univ British Columbia, Prot Engn Network Ctr Excellence, Vancouver, BC V6T 1Z1, Canada
[2] Univ British Columbia, Dept Chem, Vancouver, BC V6T 1Z1, Canada
[3] Univ York, Dept Chem, Struct Biol Lab, York YO10 5DD, N Yorkshire, England
[4] Louisiana State Univ, Dept Biol, Baton Rouge, LA 70808 USA
[5] Louisiana State Univ, Dept Chem, Baton Rouge, LA 70808 USA
关键词
D O I
10.1038/35090602
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated(3). Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by Xray diffraction. We formulate a general catalytic mechanism for all retaining beta -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.
引用
收藏
页码:835 / 838
页数:4
相关论文
共 29 条
[1]   LIFETIMES OF OXOCARBENIUM IONS IN AQUEOUS-SOLUTION FROM COMMON ION INHIBITION OF THE SOLVOLYSIS OF ALPHA-AZIDO ETHERS BY ADDED AZIDE ION [J].
AMYES, TL ;
JENCKS, WP .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1989, 111 (20) :7888-7900
[2]   THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY [J].
BAILEY, S .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1994, 50 :760-763
[3]   SOME STUDIES ON CATALYSIS BY LYSOZYME [J].
BALLARDIE, FW ;
CAPON, B ;
CUTHBERT, MW ;
DEARIE, WM .
BIOORGANIC CHEMISTRY, 1977, 6 (04) :483-509
[4]   STRUCTURE OF HEN EGG-WHITE LYSOZYME - A 3-DIMENSIONAL FOURIER SYNTHESIS AT 2A RESOLUTION [J].
BLAKE, CCF ;
KOENIG, DF ;
MAIR, GA ;
NORTH, ACT ;
PHILLIPS, DC ;
SARMA, VR .
NATURE, 1965, 206 (4986) :757-&
[5]   Crystallography & NMR system:: A new software suite for macromolecular structure determination [J].
Brunger, AT ;
Adams, PD ;
Clore, GM ;
DeLano, WL ;
Gros, P ;
Grosse-Kunstleve, RW ;
Jiang, JS ;
Kuszewski, J ;
Nilges, M ;
Pannu, NS ;
Read, RJ ;
Rice, LM ;
Simonson, T ;
Warren, GL .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1998, 54 :905-921
[6]   The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase [J].
Burmeister, WP ;
Cottaz, S ;
Driguez, H ;
Iori, R ;
Palmieri, S ;
Henrissat, B .
STRUCTURE, 1997, 5 (05) :663-675
[7]  
CHIPMAN DM, 1968, J BIOL CHEM, V243, P487
[8]   APPLICATION OF SECONDARY ALPHA-DEUTERIUM KINETIC ISOTOPE EFFECTS TO STUDIES OF ENZYME CATALYSIS . GLYCOSIDE HYDROLYSIS BY LYSOZYME AND BETA-GLUCOSIDASE [J].
DAHLQUIS.FW ;
RANDMEIR, T ;
RAFTERY, MA .
BIOCHEMISTRY, 1969, 8 (10) :4214-&
[9]  
Davies G., 1998, COMPREHENSIVE BIOL C, P119
[10]   Snapshots along an enzymatic reaction coordinate:: Analysis of a retaining β-glycoside hydrolase [J].
Davies, GJ ;
Mackenzie, L ;
Varrot, A ;
Dauter, M ;
Brzozowski, AM ;
Schülein, M ;
Withers, SG .
BIOCHEMISTRY, 1998, 37 (34) :11707-11713