Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

被引:99
作者
Horton, NC
Newberry, KJ
Perona, JJ [1 ]
机构
[1] Univ Calif Santa Barbara, Dept Chem, Santa Barbara, CA 93106 USA
[2] Univ Calif Santa Barbara, Interdept Program Biochem & Mol Biol, Santa Barbara, CA 93106 USA
关键词
D O I
10.1073/pnas.95.23.13489
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
The 2.15-Angstrom resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere mater on this metal is positioned approximately in-line for attack on the scissile phosphate, This structure corroborates the observation that the pro-S-P phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step hp a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
引用
收藏
页码:13489 / 13494
页数:6
相关论文
共 38 条
[1]  
AGGARWAL A K, 1990, Methods (Orlando), V1, P83, DOI 10.1016/S1046-2023(05)80150-1
[2]   STRUCTURE AND FUNCTION OF RESTRICTION ENDONUCLEASES [J].
AGGARWAL, AK .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 1995, 5 (01) :11-19
[3]   RAPID REACTION ANALYSIS OF THE CATALYTIC CYCLE OF THE ECORV RESTRICTION-ENDONUCLEASE [J].
BALDWIN, GS ;
VIPOND, IB ;
HALFORD, SE .
BIOCHEMISTRY, 1995, 34 (02) :705-714
[4]   FREE-ENERGY CALCULATIONS BY COMPUTER-SIMULATION [J].
BASH, PA ;
SINGH, UC ;
LANGRIDGE, R ;
KOLLMAN, PA .
SCIENCE, 1987, 236 (4801) :564-568
[5]   STRUCTURAL BASIS FOR THE 3'-5' EXONUCLEASE ACTIVITY OF ESCHERICHIA-COLI DNA-POLYMERASE-I - A 2 METAL-ION MECHANISM [J].
BEESE, LS ;
STEITZ, TA .
EMBO JOURNAL, 1991, 10 (01) :25-33
[6]   Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates [J].
Brautigam, CA ;
Steitz, TA .
JOURNAL OF MOLECULAR BIOLOGY, 1998, 277 (02) :363-377
[7]   CRYSTALLOGRAPHIC R-FACTOR REFINEMENT BY MOLECULAR-DYNAMICS [J].
BRUNGER, AT ;
KURIYAN, J ;
KARPLUS, M .
SCIENCE, 1987, 235 (4787) :458-460
[8]  
CHENG X, 1994, EMBO J, V17, P3927
[9]   CO(III) COMPLEX PROMOTED HYDROLYSIS OF PHOSPHATE DIESTERS - COMPARISON IN REACTIVITY OF RIGID CIS-DIAQUOTETRAAZACOBALT(III) COMPLEXES [J].
CHIN, J ;
BANASZCZYK, M ;
JUBIAN, V ;
ZOU, X .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1989, 111 (01) :186-190
[10]   EVIDENCE FOR THE ROLE OF SOLVATED METAL HYDROXIDE IN THE HAMMERHEAD CLEAVAGE MECHANISM [J].
DAHM, SC ;
DERRICK, WB ;
UHLENBECK, OC .
BIOCHEMISTRY, 1993, 32 (48) :13040-13045