Thermodynamic Evaluation of Binding Interactions in the Methionine Repressor System of Escherichia coli using Isothermal Titration Calorimetry

Biochemistry ◽  
1995 ◽  
Vol 34 (10) ◽  
pp. 3212-3221 ◽  
Author(s):  
David E. Hyre ◽  
Leonard D. Spicer
Keyword(s):  
Escherichia Coli ◽  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
Thermodynamic Evaluation ◽  
Binding Interactions

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

2004 ◽  
Vol 377 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Susan L. CLUGSTON ◽  
Rieko YAJIMA ◽  
John F. HONEK
Keyword(s):  
Escherichia Coli ◽  
Isothermal Titration Calorimetry ◽  
Metal Binding ◽  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Titration Calorimetry ◽  
Glyoxalase I ◽  
E Coli ◽  
Catalytic Metal ◽  
Bivalent Metals

GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn2+ but maximal activation with Ni2+. To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the kcat without significantly affecting the Km for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn2+, 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 (Ka)>107 M−1, with the exception of Mn2+ (Ka of the order of 106 M−1). One of the ligands to the catalytic metal, His5, was altered to glutamine, a side chain found in the Zn2+-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 Zn2+-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.

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