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Published online before print May 28, 2004
Protein Science, DOI: 10.1110/ps.04725204
 Copyright © 2004 The Protein Society
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Identification of active site residues in mevalonate diphosphate decarboxylase: Implications for a family of phosphotransferases

Dmitriy Krepkiy and Henry M. Miziorko

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA

(RECEIVED March 9, 2004; FINAL REVISION April 13, 2004; ACCEPTED April 13, 2004)



Abstract

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 103 and 105-fold decreases in kcat 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 Km 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, phosphomevalonate kinase (GHMP kinase) family.

Keywords: mevalonate diphosphate decarboxylase; mevalonate pyrophosphate decarboxylase; GHMP kinase family; active site mapping; general base catalyst

Abbreviations: MDD, mevalonate diphosphate decarboxylase • MK, mevalonate kinase • CDP, cytidine-5'-diphospho • GHMP, galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase • MPP, mevalonate diphosphate/pyrophosphate • IPP, isopentenyl diphosphate/pyrophosphate

Reprint requests to: Henry M. Miziorko, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA; e-mail: miziorko{at}mcw.edu; fax: (414) 456-6570.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04725204.


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