CATALYTIC ACTIVITY OF THE N-TERMINAL DOMAIN OF ESCHERICHIA-COLI ASPARAGINE-SYNTHETASE-B CAN BE REENGINEERED BY SINGLE-POINT MUTATION

The development of mechanistic strategies for the modification of enzy me function is of considerable biotechnological interest. We now repor t that replacement of the catalytically important residue Asn-74 by as partic acid (N74D) in the N-terminal domain of Escherichia coli aspara gine synthetase B (AS-B) confers nitrile hydratase activity upon the m utant enzyme. Furthermore, while wild type AS-B can efficiently cataly ze the hydrolysis of glutamine to glutamate, the N74D AS-B mutant exhi bits very low glutaminase activity. These results are consistent with similar experiments on papain, supporting the hypothesis that mutation of a critical active site residue to affect the partitioning of an in termediate common to multiple reaction mechanisms may represent an app roach by which enzymes can be obtained with different catalytic functi on. Our experiments also provide the first direct chemical evidence fo r a close mechanistic relationship between papain, a thiol protease, a nd AS-B, a class II Ntn amidotransferase. These enzymes are not likely to have arisen by evolution from a common ancestral protein.