ACTIVE-SITE MUTAGENESIS OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE - REPLACEMENT OF SERINE-102 WITH NONNUCLEOPHILIC AMINO-ACIDS

被引:31
作者
BUTLERRANSOHOFF, JE
ROKITA, SE
KENDALL, DA
BANZON, JA
CARANO, KS
KAISER, ET
MATLIN, AR
机构
[1] OBERLIN COLL,DEPT CHEM,OBERLIN,OH 44074
[2] ROCKEFELLER UNIV,BIOORGAN CHEM & BIOCHEM LAB,NEW YORK,NY 10021
关键词
D O I
10.1021/jo00027a027
中图分类号
O62 [有机化学];
学科分类号
070303 ; 081704 ;
摘要
The active-site nucleophile in bacterial alkaline phosphatase, Ser-102, was replaced using site-directed mutagenesis, with leucine and, separately, alanine. Remarkably, the mutant enzymes S102A and S102L, which lack a nucleophilic side chain at position 102, still catalyze the hydrolysis of phosphate monoesters. The k(cat) values are, respectively, approximately 1/1000 and approximately 1/500 of the wild-type enzyme. The mutant enzymes have K(m) values, K(i) values (inorganic phosphate), and pH profiles that are similar to wild-type. Several experiments argue against the possibility of wild-type contamination. The S102L mutant has a substrate-dependent partition ratio which is consistent with a change in the enzyme mechanism. Direct hydrolysis by zinc-activated water may account for the catalytic activity of these mutant enzymes. This work illustrates the use of site-directed mutagenesis to uncover latent enzyme activity and reinforces the idea that the zinc atoms in the active site are the critical structural feature in the design of alkaline phosphatase.
引用
收藏
页码:142 / 145
页数:4
相关论文
共 30 条
  • [1] AAVIKSSAR A, UNPUB
  • [2] Ames BN., 1966, METHOD ENZYMOL, V8, P115, DOI DOI 10.1016/0076-6879(66)08014-5
  • [3] EVIDENCE FOR A PHOSPHORYL-ENZYME INTERMEDIATE IN ALKALINE PHOSPHATASE CATALYZED REACTIONS
    BARRETT, H
    BUTLER, R
    WILSON, IB
    [J]. BIOCHEMISTRY, 1969, 8 (03) : 1042 - &
  • [4] DEOXYNUCLEOSIDE PHOSPHORAMIDITES - A NEW CLASS OF KEY INTERMEDIATES FOR DEOXYPOLYNUCLEOTIDE SYNTHESIS
    BEAUCAGE, SL
    CARUTHERS, MH
    [J]. TETRAHEDRON LETTERS, 1981, 22 (20) : 1859 - 1862
  • [5] AMINO-ACID-SEQUENCE OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE
    BRADSHAW, RA
    CANCEDDA, F
    ERICSSON, LH
    NEUMANN, PA
    PICCOLI, SP
    SCHLESINGER, MJ
    SHRIEFER, K
    WALSH, KA
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1981, 78 (06): : 3473 - 3477
  • [6] USE OF SITE-DIRECTED MUTAGENESIS TO ELUCIDATE THE ROLE OF ARGININE-166 IN THE CATALYTIC MECHANISM OF ALKALINE-PHOSPHATASE
    BUTLERRANSOHOFF, JE
    KENDALL, DA
    KAISER, ET
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (12) : 4276 - 4278
  • [7] STEREOCHEMISTRY OF PHOSPHO GROUP TRANSFER CATALYZED BY A MUTANT ALKALINE-PHOSPHATASE
    BUTLERRANSOHOFF, JE
    KENDALL, DA
    FREEMAN, S
    KNOWLES, JR
    KAISER, ET
    [J]. BIOCHEMISTRY, 1988, 27 (13) : 4777 - 4780
  • [8] BUTLERRANSOHOFF JE, 1989, MET IONS BIOL SYST, V25, P395
  • [9] FUNCTION OF ARGININE-166 IN THE ACTIVE-SITE OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE
    CHAIDAROGLOU, A
    BREZINSKI, DJ
    MIDDLETON, SA
    KANTROWITZ, ER
    [J]. BIOCHEMISTRY, 1988, 27 (22) : 8338 - 8343
  • [10] COLEMAN JE, 1983, ADV ENZYMOL RAMB, V55, P381