Tyrosine 222, a member of the YXDD motif of MuLV RT, is catalytically essential and is a major component of the fidelity center

Tyrosine 222 of MuLV RT is an invariant residue of the highly conserved YXD D motif in the reverse transcriptase class of enzymes. The residue X is Met 184 in HIV-I RT and Val 223 in MuLV RT. This residue has been implicated i n the fidelity of DNA synthesis, whereas the role of the preceding tyrosine in this aspect, as well as in the catalytic mechanism of MuLV RT, remains to be elucidated. We have substituted Tyr 222 with Phe, Ser, and Ala by sit e-directed mutagenesis and have characterized the properties of the individ ual mutant enzymes. The results show that Tyr-->Phe substitution did not af fect the polymerase activity of the enzyme, while Tyr-->Ser and Tyr-->Ala s ubstitutions significantly reduced the polymerase activity. The pyrophospho rolysis activities of these mutants showed the same trend as the polymerase activities, suggesting an essential role for Y222 in the catalytic mechani sm of MuLV RT. One of the most interesting observations of Y-->F substituti on was the significantly increased fidelity of DNA synthesis on RNA templat es. In addition, a limited extent of ribonucleotide incorporation on RNA te mplate that was consistently noted with the wild-type enzyme was reduced wi th the Y222F mutant. The resistance to all four ddNTPs, however, persisted in the wild type and Y222 mutants on the RNA template. A ternary complex mo del of MuLV RT shows that (a) the aromatic ring of Tyr/Phe is positioned be tween the terminal and penultimate primer bases and (b) the phenolic OH gro up is seen within hydrogen bonding distance with the base moieties of two t emplate and penultimate primer nucleotides. We propose that the base stacki ng interaction of Tyr 222 stabilizes the primer terminus position which is essential for the catalytic reaction. However, the weaker stacking interact ion of Y compared to F, due to polarization of the pi-charge toward the phe noxyl-OH as well as the resonating character of its H-bond center, may prov ide slight flexibility to the position of the template base which may be re sponsible for the error-proneness of MuLV RT.