Linked Life Data

14871497

Source:http://linkedlifedata.com/resource/pubmed/id/14871497

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2-3
pubmed:dateCreated
2004-2-11
pubmed:abstractText
Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-3002
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
1608
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
190-9
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase.
pubmed:affiliation
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't