Improving the catalytic activity of a thermophilic enzyme at low temperatures

Enzymes from thermophilic organisms often are barely active at low temperat ures. To obtain a better understanding of this sluggishness, we used DNA sh uffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants produ cing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid ch anges and combinations of these changes improved growth appreciably. Five s ingly and doubly altered protein variants with changes at the N- and C-term ini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified var iant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait, Although the affinities for bot h the substrate and the product decreased appreciably in most protein varia nts, these defects were offset by the accumulation of high levels of the en zyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temp eratures is limited by the dissociation of the enzyme-product complex. In c ontrast, representative protein variants bind and release the product far m ore rapidly, shifting the bottleneck to the preceding chemical step. The tu rnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activit y at low temperatures. In support of this interpretation, the rate of tryps inolysis or of thermal denaturation is accelerated significantly in the act ivated protein variants.