Linked Life Data

10727235

Source:http://linkedlifedata.com/resource/pubmed/id/10727235

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2000-5-4
pubmed:abstractText
The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/1,2-dihexanoic-sn-glycero-3-phosphos..., http://linkedlifedata.com/resource/pubmed/chemical/1,2-dihexanoylphosphatidylethanolami..., http://linkedlifedata.com/resource/pubmed/chemical/Choline, http://linkedlifedata.com/resource/pubmed/chemical/Glutamic Acid, http://linkedlifedata.com/resource/pubmed/chemical/Phenylalanine, http://linkedlifedata.com/resource/pubmed/chemical/Phosphatidylcholines, http://linkedlifedata.com/resource/pubmed/chemical/Phosphatidylethanolamines, http://linkedlifedata.com/resource/pubmed/chemical/Phosphatidylserines, http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Type C Phospholipases, http://linkedlifedata.com/resource/pubmed/chemical/Tyrosine, http://linkedlifedata.com/resource/pubmed/chemical/phosphatidylcholine-specific...
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
39
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3410-5
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity.
pubmed:affiliation
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA. sfmartin@mail.utexas.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't