Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA)

The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism, Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K_M^tRNA = 1.9 ± 0.7 μM and K_M^AdoMet 98 ± 5.0 μM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ₁-tRNA^Tyr, with K_i values of 133 ± 18 and 4.6 ± 0.5 μM for sinefungin and S- adenosylhomocysteine, respectively. Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ₁-tRNA^Tyr when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA^Tyr) was competitive and noncompetitive against the substrates preQ₁-tRNA^Tyr and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ₁-tRNA^Tyr, but noncompetitive against AdoMet. However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive. Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ₁-tRNA^Tyr binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA. The chemical mechanism that we previously proposed for the QueA-catalyzed reaction [Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D. (2000) Org. Lett. 2, 1307-1310] is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme.