Linked Life Data

8664255

Source:http://linkedlifedata.com/resource/pubmed/id/8664255

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
15
pubmed:dateCreated
1996-8-6
pubmed:abstractText
Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase). Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase. In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration. Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant. By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4662-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary structure and catalytic properties.
pubmed:affiliation
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't