Mutagenesis of rat liver arginase expressed in Escherichia coli: role of conserved histidines.
Rat liver arginase has been overexpressed in Escherichia coli using a T7-based expression system. The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme. The recombinant wild-type protein contains six Mn(II) ions per trimer, in good agreement with results obtained with the fully active native enzyme. However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer even after extensive dialysis. Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly conserved in arginases from evolutionarily divergent species. The replacement of His101 and His126 with Asn by site-directed mutagenesis produced only modest effects on enzymatic activity when measured in the presence of Mn(II) ions. However, EDTA treatment of these mutant enzymes reduced activity to < 0.2% of that for the wild-type enzyme. The activity of wild-type enzyme and the His141 Asn mutant was unaffected by treatment with EDTA. Thus, His101 and His126 are proposed to be ligands to the binuclear Mn(II) center of the enzyme. The His141 Asn mutation produced an enzyme which, in contrast to the native, wild-type, His101 Asn, and His126 Asn arginases, was not inactivated by diethyl pyrocarbonate. These results suggest a catalytic role for His141.
Cavalli
R C
RC
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Burke
C J
CJ
Kawamoto
S
S
Soprano
D R
DR
Ash
D E
DE
D000818:Animals; D001119:Arginase / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008345:Manganese; D006639:Histidine; D004492:Edetic Acid; D001119:Arginase; D004047:Diethyl Pyrocarbonate