Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease.
To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.
Sperka
Tamás
T
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen H-4010 Debrecen, Egyetem tér 1, PO Box 6, Hungary.
Boross
Péter
P
Eizert
Helga
H
Tözsér
József
J
Bagossi
Péter
P
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D019281:Dimerization; D004795:Enzyme Stability; D016333:HIV Protease / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D016092:Spumavirus / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease