Mechanistic studies of the reaction catalyzed by eubacterial tRNA -guanine transglycosylase.

Abstract

The modified base queuine (Q) is found at the wobble position of tRNAs with anticodon sequences of GUN. In eubacteria a Q-precursor, preQ₁, is exchanged for guanine-34 by tRNA-guanine transglycosylase (TGT). Further modification at position 34 leads to Q-tRNA. Relatively little is known about the factors that influence the initiation and control of the TGT-catalyzed reaction. The kinetic mechanism of TGT was determined using initial rate kinetics and competitive inhibition studies. TGT was found to proceed through a ping-pong mechanism, with tRNA binding first. To facilitate our understanding of the chemical mechanism, the role of an active site cysteine (145 in <italic>E. coli</italic>) was investigated. A series of mutants was generated to probe the effects of charge and hydrogen-bonding potential at this position. The C145A and C145S mutants each showed a moderate (ca. 10-fold) increase in <italic>k_cat</italic>, with corresponding increases in the <italic>K_M</italic>s for both substrates. Alternatively, the aspartic acid mutant did not significantly alter the catalytic rate, but did cause 20- and 200-fold increases in the tRNA and guanine <italic>K_M</italic>s, respectively. To better understand the Significance of the kinetic studies, the pH-dependence of the TGT reaction was determined. The wild-type and mutant enzymes Showed bell-shaped pH profiles. Although the wild-type and mutant enzymes differ in pH optimum, this alone does not account for the altered activity seen with the mutants. Finally, the binding of both substrates was investigated. No difference in binding was observed between the wild-type and mutant TGTs for either G(34)-tRNA or preQ1(34)-tRNA. The binding of guanine was estimated through the use of a known competitive inhibitor (7-methylguanine). The <italic>K_i</italic> values for C145A and C145S were ca. 100- and 500-fold greater than WT, respectively. The <italic>K_i</italic> for C145D was beyond the limits of 7-methylguanine solubility. The results of this work support the hypothesis that Cys145 is involved in heterocycle binding and specificity. Both the rate enhancements, and the increases in <italic>K_M</italic> for the C145A and C145S mutants are consistent with product (guanine) release being partially rate-limiting. The loss of the cysteine functionality weakens the interaction with guanine and allows faster dissociation of the base following bond cleavage.