Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies

GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn(2+) but maximal activation with Ni(2+). To elucidate further the metal site in E. coli GlxI, several approaches were undertake...

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Bibliographic Details
Published in:Biochemical journal 2004-01, Vol.377 (Pt 2), p.309-316
Main Authors: Clugston, Susan L, Yajima, Rieko, Honek, John F
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Binding Sites
Calorimetry
Enzyme Activation
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Kinetics
Lactoylglutathione Lyase - chemistry
Lactoylglutathione Lyase - genetics
Lactoylglutathione Lyase - metabolism
Metals - metabolism
Mutagenesis, Site-Directed
Thermodynamics
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Summary:GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn(2+) but maximal activation with Ni(2+). To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the k (cat) without significantly affecting the K (m) for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn(2+), which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant ( K (a))>10(7) M(-1), with the exception of Mn(2+) ( K (a) of the order of 10(6) M(-1)). One of the ligands to the catalytic metal, His(5), was altered to glutamine, a side chain found in the Zn(2+)-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn(2+)-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj20030271