Document Detail

Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases.
MedLine Citation:
PMID:  15169949     Owner:  NLM     Status:  MEDLINE    
A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.
Dmitriy Krepkiy; Henry M Miziorko
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2004-05-28
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  13     ISSN:  0961-8368     ISO Abbreviation:  Protein Sci.     Publication Date:  2004 Jul 
Date Detail:
Created Date:  2004-06-24     Completed Date:  2005-01-25     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1875-81     Citation Subset:  IM    
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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MeSH Terms
Adenosine Triphosphate / analogs & derivatives,  chemistry
Amino Acid Sequence
Amino Acid Substitution*
Aspartic Acid / genetics
Binding Sites / genetics
Carboxy-Lyases / chemistry*,  genetics
Lysine / genetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphotransferases / chemistry
Point Mutation*
Saccharomyces cerevisiae / enzymology*
Saccharomyces cerevisiae Proteins / chemistry*,  genetics
Sequence Alignment
Substrate Specificity / genetics
Grant Support
Reg. No./Substance:
0/Saccharomyces cerevisiae Proteins; 56-65-5/Adenosine Triphosphate; 56-84-8/Aspartic Acid; 56-87-1/Lysine; EC 2.7.-/Phosphotransferases; EC 4.1.1.-/Carboxy-Lyases; EC decarboxylase

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