Analysis of stability and catalytic properties of two tryptophanases from a thermophile

Two tryptophanases, Tna1 and Tna2, both of which were cloned from the therm ophile Symbiobacterium thermophilum, differ in their enzymatic properties, such as thermal stability, catalytic efficiency and activation energy of ca talysis, despite the great similarity (92%) in their amino acid sequences. Chimeric tryptophanases were constructed by recombination of the two genes to try to elucidate the molecular basis for the difference. The stability o f each chimeric enzyme was roughly proportional to the content of amino aci d residues from Tna1, Three regions, tentatively named regions 2, 4 and 5, which contained the amino acid residues 70-129, 192-298 and 299-453, respec tively, were especially important for the increase in thermal stability. Si te-directed mutagenesis revealed that V104 in region 2 and Y198 in region 4 of Tna1 were involved in the increase in thermal stability of Tna1, Amino acid residues contributing to the higher catalytic efficiency of Tna1 were similarly analyzed, using the chimeric tryptophanases, and found to be loca ted in region 5, Site-directed mutagenesis revealed that I383 and G395 in T na1, which were presumably located close to the putative active center, pla yed an active role in the increase of catalytic efficiency of Tna1, The act ivation energy of catalysis was proportional to the content of amino acid r esidues from Tna2, suggesting the amino acid residues responsible for the d ifference were dispersed over the whole molecule.