Structure - activity relationship of pyridoxalphosphate dependent enzymes

Ornithine aminotransferase (OAT) catalyzes the transfer of the amino-group from l-ornithine to α-ketoglutarate, forming l-glutamate and glutamate-5-semialdehyde. It is found in bacteria, fungi, plants and higher animals including man. The structure of the human enzyme, both free [1] and with irreversibly-bound substrate analogues [2, 3], has been solved by X-ray crystallography. There are many similarities in structure, particularly at the active site, with other members of the a­family of pyridoxal-5'-phosphate-dependent enzymes. This is especially true for GABA-aminotransferase, the active site of which contains only two significant differences from that of OAT [4]. Unlike majority of aminotransferases, both OAT and GABA-AT catalyze the transfer of amino group from carbon atom that does not bear carboxyl group. However, both enzymes are using α-ketoglutarate to complete the transamination cycle and they are able to transfer of the amino group from glutamate. To explain the part played by the amino acid residues from active site, we used mutation analysis. Two sets of mutants were prepared, the first one based on comparison of active sites of GABA-AT and OAT and the second based on comparison of OAT and aminotransferases catalyzing transfer of amino group exclusively from carbon bearing carboxyl group.The specific activity towards l-ornithine and GABA and rate constants for single steps of the reactions of mutants were compared with those of the wild type OAT. "www.biomikro.vscht.cz":[ http://biomikro.vscht.cz/groups/lab211/oat.html]

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