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, His(91), His(110), His(162) and His(178), 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 His(91) or His(162) for asparagine resulted in complete loss of activity. The His(110) enzyme had a reduced V-max and the His(178) 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 His(91) to Gln(91) observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins. (C) 1995 Academic Press Limited