Journal
PROCESS BIOCHEMISTRY
Volume 45, Issue 4, Pages 607-612Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2009.12.006
Keywords
Prephenate dehydrogenase; Streptococcus mutans; MALDI-TOF; Size exclusion chromatography; Site-directed mutagenesis; Tyrosine
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Funding
- Korea Research Institute of Standards and Science
- Korea Science and Engineering Foundation
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Streptococcus mutans is commonly found in the human oral cavity. Functionally active prephenate dehydrogenase (PDH) catalyzes conversion of prephenate to 4-hydroxylphenylpyruvate. In order to characterize S. mutans-PDH (Sm-PDH), a pdh gene was expressed using Escherichia coli expression vector PET-15b and Sm-PDH was affinity purified using a nickel column. The molecular weight of this enzyme was determined using MALDI-TOF and size exclusion chromatography. PDH assays were employed to determine the kinetic parameters of the reaction catalyzed by the purified enzyme and to evaluate the effects of temperature, pH and ion concentration on PDH activity. Maximum PDH activity was obtained at 37 degrees C and pH 6.8 or below. Cations had little or no effect on PDH activity. Site-directed mutations were introduced into pdh. Amino acid replacements at conserved residues markedly reduced or eliminated enzyme activity. L-Tyrosine, a feedback inhibitor of PDH, showed strong inhibitory effects on PDH activity. (C) 2009 Elsevier Ltd. All rights reserved.
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