CYSTEINE-265 IS IN THE ACTIVE-SITE OF, BUT IS NOT ESSENTIAL FOR CATALYSIS BY TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE (TGT) FROM ESCHERICHIA-COLI

Site-directed mutagenesis and X-ray absorption spectroscopy studies ha ve previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coil is a zinc metalloprotein and identified the enzymic l igands to the zinc [Chong et al. (1995), Biochemistry 34, 3694-3701; G arcia et al. (1966), Biochemistry 35, 3133-3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower s pecific activity, but was not found to be involved in the zinc site. T he present report demonstrates that TGT is inactivated by treatment wi th thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, t his inactivation is shown to be due to modification of cysteine 265. T he kinetic parameters for the mutants TGT (C265A) and TGT (C265S), how ever, suggest that this residue is not performing a critical role in t he TGT reaction. We conclude that cysteine 265 is in the active site o f TGT, but is not performing a critical catalytic function. This concl usion is supported by the recent determination of the X-ray crystal st ructure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850-2857], which reveals that the residue corresponding to cys teine 265 is distant from the putative catalytic site, but is in the m iddle of a region of the enzyme surface proposed to bind tRNA.