ESCHERICHIA-COLI K-12 URIDINE PHOSPHORYLASE PROTEIN ENGINEERING - THEROLE OF CYSTEINE RESIDUES IN ENZYME FUNCTIONING

Site-directed mutagenesis enabled construction of several mutant Esche richia coli uridine phosphorylase (UPH) varieties with point substitut ions of cysteine residues and scanning mutations in the polypeptide ch ain region adjacent to Cys-136. Studying expression of udp gene with r eplaced cysteine codons, we demonstrated that this protein has neither intra- nor intermolecular disulfide bonds. Estimating the enzymic act ivity of UPH mutants, we found that Cys-65 is not involved in protein functioning. On the other hand, Cys-206 seems to be involved in stabil ization of UPH active quaternary structure. Strong evidence exists tha t Cys-136 plays a central structural-functional role and participates in the formation of the active form of UPH.