The mutability of enzyme active-site shape determinants.
Investigations of enzyme action typically focus on elucidating the catalytic roles of hydrogen bonding interactions between polar active-site residues and substrate molecules. Less clear is the importance of non-hydrogen bonding contacts to enzymatic rate accelerations. To investigate the importance of such interactions in a model system, six residues that participate in van der Waals contacts with substrate glucose within the active site of Escherichia coli glucokinase were individually randomized via site-directed mutagenesis. In vivo selection in a glucokinase-deficient bacterium was employed to identify amino acid substitutions that were complicit with enzyme activity. The results suggest that small residues, such as alanine and glycine, are largely immutable, whereas larger amino acids are more tolerant of diverse substitution patterns. Surprisingly, a glucokinase variant that contains glycine in place of six non-hydrogen bonding contacts retains approximately 1% of the wild-type activity. These findings establish non-hydrogen bonding shape determinants as highly appealing targets for widespread substitution during efforts to redesign the catalytic properties of natural enzymes.
Miller
Brian G
BG
Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306-4390, USA. miller@chem.fsu.edu
D019943:Amino Acid Substitution; D001665:Binding Sites; D018274:Electroporation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D014644:Genetic Variation; D005941:Glucokinase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012641:Selection, Genetic; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005941:Glucokinase; D005947:Glucose; D005998:Glycine