Exploring the role of histidines in the catalytic activity of duck delta-crystallins using site-directed mutagenesis.
The duck delta 2-crystallin gene encodes an enzymatically-active argininosuccinate lyase while the delta 1-crystallin gene product, although 94% identical, is enzymatically inactive. Four histidine residues in the duck delta 2-crystallin. His91, His110, His162 and His178, were converted to asparagine residues in an effort to define the role of histidines in the catalytic process of this enzyme-crystallin and to explain the lack of enzyme activity in the delta 1-crystallin protein. The recombinant mutant proteins were expressed in E. coli and purified to homogeneity for analysis. These four residues were chosen because they fall within highly conserved regions of argininosuccinate lyases from several species. This analysis revealed that change of His91 or His162 for asparagine resulted in complete loss of activity. The His110 enzyme had a reduced Vmax and the His178 enzyme was near normal in its kinetic properties. These data confirm the roles of histidine in the catalytic process of this enzyme-crystallin and suggest that the change of His91 to Gln91 observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins.
Patejunas
G
G
Department of Molecular and human genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Barbosa
P
P
Lacombe
M
M
O'Brien
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002645:Chickens / Q000378:metabolism; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D004372:Ducks / Q000378:metabolism*; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013045:Species Specificity
D003459:Crystallins; D011994:Recombinant Proteins; D006639:Histidine