Interaction of two arginine residues in lactate oxidase with the enzyme flavin: conversion of FMN to 8-formyl-FMN.
Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of alpha-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH(3)- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.
Yorita
K
K
Institute for Enzyme Research, University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan.
Matsuoka
T
T
Misaki
H
H
Massey
V
V
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D017124:Conserved Sequence; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013289:Streptococcaceae / Q000201:enzymology*; D013816:Thermodynamics
C417272:8-formylflavin mononucleotide; D005420:Flavoproteins; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D001120:Arginine; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase