PMID- 10231536
OWN - NLM
STAT- MEDLINE
DCOM- 19990601
LR  - 20161124
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 18
DP  - 1999 May 4
TI  - Mutagenesis of two conserved tryptophan residues of the E-type ATPases:
      inactivation and conversion of an ecto-apyrase to an ecto-NTPase.
PG  - 5849-57
AB  - A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed
      mutagenesis to assess the functional significance of two highly conserved
      tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in 
      nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly
      expressed ecto-apyrase completely devoid of nucleotidase activity.
      Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular
      distribution clearly different from that of the wild-type enzyme, with the
      majority of the immunoreactivity concentrated in the interior of the cell. Unlike
      the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron
      Blue and was sensitive to proteolytic digestion by chymotrypsin. These results
      suggest alteration of the tertiary structure, causing the enzyme to be improperly
      folded and retained within the cell. In contrast, mutation of tryptophan 459 to
      alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished
      NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a
      cellular pattern similar to that of the wild-type enzyme, distributed along the
      cell periphery and in cell processes. Coupling this active W459A mutation to a
      previously described mutation (D219E) resulted in an enzyme which preferentially 
      hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double
      mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In 
      addition, the double mutant is substantially less sensitive to inhibition by
      azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation
      of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase 
      to an ecto-NTPase.
FAU - Smith, T M
AU  - Smith TM
AD  - Department of Pharmacology and Cell Biophysics, College of Medicine, University
      of Cincinnati, Ohio 45267-0575, USA.
FAU - Lewis Carl, S A
AU  - Lewis Carl SA
FAU - Kirley, T L
AU  - Kirley TL
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Adenine Nucleotides)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Triazines)
RN  - 30KYC7MIAI (Aspartic Acid)
RN  - 3KX376GY7L (Glutamic Acid)
RN  - 5DV0L8V99J (Cibacron Blue F 3GA)
RN  - 8DUH1N11BX (Tryptophan)
RN  - 968JJ8C9DV (Sodium Azide)
RN  - EC 3.4.21.1 (Chymotrypsin)
RN  - EC 3.6.- (Acid Anhydride Hydrolases)
RN  - EC 3.6.1.- (Adenosine Triphosphatases)
RN  - EC 3.6.1.- (ectoATPase)
RN  - EC 3.6.1.15 (Nucleoside-Triphosphatase)
RN  - OF5P57N2ZX (Alanine)
SB  - IM
MH  - Acid Anhydride Hydrolases/*metabolism
MH  - Adenine Nucleotides/metabolism
MH  - Adenosine Triphosphatases/*antagonists & inhibitors/chemistry/genetics/metabolism
MH  - Alanine/genetics
MH  - Amino Acid Sequence
MH  - Animals
MH  - Aspartic Acid/genetics
MH  - Brain/enzymology
MH  - COS Cells
MH  - Chymotrypsin/metabolism
MH  - Conserved Sequence/*genetics
MH  - Enzyme Inhibitors/pharmacology
MH  - Escherichia coli/enzymology/genetics
MH  - Glutamic Acid/genetics
MH  - Humans
MH  - Hydrolysis
MH  - Molecular Sequence Data
MH  - *Mutagenesis, Site-Directed
MH  - Nucleoside-Triphosphatase
MH  - Protein Folding
MH  - Sodium Azide/pharmacology
MH  - Transfection
MH  - Triazines/pharmacology
MH  - Tryptophan/*genetics
EDAT- 1999/05/08 00:00
MHDA- 1999/05/08 00:01
CRDT- 1999/05/08 00:00
PHST- 1999/05/08 00:00 [pubmed]
PHST- 1999/05/08 00:01 [medline]
PHST- 1999/05/08 00:00 [entrez]
AID - 10.1021/bi990171k [doi]
AID - bi990171k [pii]
PST - ppublish
SO  - Biochemistry. 1999 May 4;38(18):5849-57. doi: 10.1021/bi990171k.