Structural characterization of human aryl sulphotransferases

被引:31
作者
Brix, LA
Duggleby, RG
Gaedigk, A
McManus, ME [1 ]
机构
[1] Univ Queensland, Dept Physiol & Pharmacol, Brisbane, Qld 4072, Australia
[2] Univ Queensland, Dept Biochem, Brisbane, Qld 4072, Australia
[3] Childrens Mercy Hosp, Dept Clin Pharmacol & Toxicol, Kansas City, MO 64108 USA
关键词
chimaeric protein; dopamine; enzyme kinetics; mutagenesis; p-nitrophenol;
D O I
10.1042/0264-6021:3370337
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
Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44-107) and B (amino acids 132-164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated K-i values towards the co-substrate 3'-phosphoadenosine 5'-phosphosulphate. and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST 1 mutant was constructed to express a protein with four amino acids deleted (P87-P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.
引用
收藏
页码:337 / 343
页数:7
相关论文
共 32 条
  • [21] Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes
    Tamura, H
    Morioka, Y
    Homma, H
    Matsui, M
    [J]. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1997, 341 (02) : 309 - 314
  • [22] ELECTROPHORETIC TRANSFER OF PROTEINS FROM POLYACRYLAMIDE GELS TO NITROCELLULOSE SHEETS - PROCEDURE AND SOME APPLICATIONS
    TOWBIN, H
    STAEHELIN, T
    GORDON, J
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1979, 76 (09) : 4350 - 4354
  • [23] Biochemistry and molecular biology of plant sulfotransferases
    Varin, L
    Marsolais, F
    Richard, M
    Rouleau, M
    [J]. FASEB JOURNAL, 1997, 11 (07) : 517 - 525
  • [24] CHIMERIC FLAVONOL SULFOTRANSFERASES DEFINE A DOMAIN RESPONSIBLE FOR SUBSTRATE AND POSITION SPECIFICITIES
    VARIN, L
    MARSOLAIS, F
    BRISSON, N
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (21) : 12498 - 12502
  • [25] VARIN L, 1992, J BIOL CHEM, V267, P1858
  • [26] FUNCTIONAL-CHARACTERIZATION OF 2 HUMAN SULFOTRANSFERASE CDNAS THAT ENCODE MONOAMINE-SULFATING AND PHENOL-SULFATING FORMS OF PHENOL SULFOTRANSFERASE - SUBSTRATE KINETICS, THERMAL-STABILITY AND INHIBITOR-SENSITIVITY STUDIES
    VERONESE, ME
    BURGESS, W
    ZHU, XY
    MCMANUS, ME
    [J]. BIOCHEMICAL JOURNAL, 1994, 302 : 497 - 502
  • [27] WEINSHILBOUM R, 1994, HDB EXPT PHARM CONJU, V112, P45
  • [28] Sulfotransferase molecular biology: cDNAs and genes
    Weinshilboum, RM
    Otterness, DM
    Aksoy, IA
    Wood, TC
    Her, C
    Raftogianis, RB
    [J]. FASEB JOURNAL, 1997, 11 (01) : 3 - 14
  • [29] Sulfuryl transfer: The catalytic mechanism of human estrogen sulfotransferase
    Zhang, HP
    Varmalova, O
    Vargas, FM
    Falany, CN
    Leyh, TS
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (18) : 10888 - 10892
  • [30] cDNA cloning and expression of a new form of human aryl sulfotransferase
    Zhu, XY
    Veronese, ME
    Iocco, P
    Mcmanus, ME
    [J]. INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, 1996, 28 (05) : 565 - 571