PMID- 10788459
OWN - NLM
STAT- MEDLINE
DCOM- 20000601
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 275
IP  - 18
DP  - 2000 May 5
TI  - Mutational analysis of the substrate binding/catalytic domains of human M form 
      and P form phenol sulfotransferases.
PG  - 13460-4
AB  - Human monoamine (M) form and simple phenol (P) form phenol sulfotransferases 
      (PSTs) are greater than 93% identical in their primary sequences and yet display 
      distinct substrate specificities and other enzymatic properties. Through the 
      generation and characterization of a series of chimeric PSTs, we have previously 
      demonstrated two highly variable regions within their sequences to be responsible 
      for determining their substrate phenotypes (Sakakibara, Y., Takami, Y., Nakayama, 
      T., Suiko, M., and Liu, M.-C. (1998) J. Biol. Chem. 273, 6242-6247). By employing 
      the site-directed mutagenesis technique, the present study aims to identify and 
      quantitatively evaluate the specific amino acid residues critical to the 
      substrate binding and catalysis in these two enzymes. Twelve mutated M-PSTs and 
      seven mutated P-PSTs were generated, expressed, and purified. Enzymatic 
      characterization showed that, of the twelve mutated M-PSTs, mutations at residues 
      Asp-86, Glu-89, and Glu-146 resulted in a dramatic decrease in V(max)/K(m) with 
      dopamine as substrate, being greater than 450 times for the D86A/E89I/E146A 
      mutated M-PST. With p-nitrophenol as substrate, the V(max)/K(m) determined for 
      the D86A/E89I/E146A-mutated M-PST increased more than 25 times and approached 
      that determined for the wild-type P-PST. These results indicated that the 
      concerted action of the three mutated residues (D86A, E89I, and E146A) is 
      sufficient for the conversion of the substrate phenotype of M-PST to that of 
      P-PST. Among the mutated P-PSTs, the I89E- and A146E-mutated P-PSTs displayed 
      considerable deviations in V(max)/K(m) with dopamine or p-nitrophenol as 
      substrate. No corresponding changes, however, were detected with the opposite 
      compound as substrate. These results indicated that, in contrast to M-PST, 
      mutations at Ala-86, Ile-89, and Ala-146 to the corresponding residues in M-PST 
      are not sufficient for rendering the change of P-PST substrate phenotype to that 
      of M-PST. For both M-PSTs and P-PSTs, mutations at Lys-48 or His-108 led to the 
      loss of sulfotransferase activities, indicating their importance in the catalytic 
      mechanism.
FAU - Liu, M C
AU  - Liu MC
AD  - Biomedical Research Center, The University of Texas Health Center at Tyler, 
      Tyler, Texas 75708, USA. liu@uthct.edu
FAU - Suiko, M
AU  - Suiko M
FAU - Sakakibara, Y
AU  - Sakakibara Y
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Isoenzymes)
RN  - EC 2.8.2.1 (Arylsulfotransferase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Arylsulfotransferase/*chemistry/genetics
MH  - Base Sequence
MH  - Binding Sites/genetics
MH  - DNA Mutational Analysis
MH  - Humans
MH  - Isoenzymes/chemistry/genetics
MH  - Molecular Sequence Data
MH  - Sequence Alignment
MH  - Substrate Specificity/genetics
EDAT- 2000/05/02 09:00
MHDA- 2000/06/03 09:00
CRDT- 2000/05/02 09:00
PHST- 2000/05/02 09:00 [pubmed]
PHST- 2000/06/03 09:00 [medline]
PHST- 2000/05/02 09:00 [entrez]
AID - S0021-9258(19)80710-X [pii]
AID - 10.1074/jbc.275.18.13460 [doi]
PST - ppublish
SO  - J Biol Chem. 2000 May 5;275(18):13460-4. doi: 10.1074/jbc.275.18.13460.