Substrate specificity of branched chain amino acid aminotransferases : The substitution of glycine to serine in the active site determines the substrate specificity for α-ketoglutarate

A branched chain aminotransferase from Thermoproteus tenax has been
identi fi ed, cloned, over-expressed and biochemically characterised. A
molecular modelling approach has been used to predict the 3D structure
allowing its comparison with other related enzymes. This enzyme has high
similarity to a previously characterised aminotransferase from Thermoproteus
uzoniensis however its substrate speci fi city shows key differences towards the
substrate α -ketoglutarate. Examination of the active sites of the two related
enzymes reveals a single amino acid substitution of a glycine residue to a serine
residue which could be responsible for this difference. When Gly104 in T. tenax
was mutated to a serine residue and the resultant enzyme characterised, this
single amino acid change resulted in a dramatic reduction in activity towards α -
ketoglutarate with an 18-fold reduction in Vmax and a 20-fold Km increase,
resulting in a 370-fold lower catalytic ef fi ciency. Structural comparisons
between the two related Thermoproteus enzymes and another branched
chain aminotransferase from Geoglobus acetivorans has revealed that the
serine residue affects the fl exibility of a key loop involved in catalysis. This
subtle difference has provided further insight into our understanding of the
substrate speci fi city of these industrially important enzymes.


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