Tryptophanless recombinant horseradish peroxidase: stability and catalytic properties.
The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.
Gazaryan
I G
IG
Chemical Faculty, Moscow State University, Moscow, 119899 GSP, Russia. igazaryan@hotmail.com
Chubar
T A
TA
Ignatenko
O V
OV
Mareeva
E A
EA
Orlova
M A
MA
Kapeliuch
Y L
YL
Savitsky
P A
PA
Rojkova
A M
AM
Tishkov
V I
VI
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D004583:Electrons; D004789:Enzyme Activation / Q000528:radiation effects; D004795:Enzyme Stability / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies; D008163:Luminescent Measurements; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase