A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes.
His15 is a conserved amino acid residue in all known class Mu glutathione transferases. This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity. The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli. Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized. The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC. For all the mutant forms except H15N, the sp. act. with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity. None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme. The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base. The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation. A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.
Widersten
M
M
Department of Biochemistry, Uppsala University, Sweden.
Mannervik
B
B
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics*; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D006639:Histidine; D005982:Glutathione Transferase