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 microM and K (M)(AdoMet)= 98 +/- 5.0 microM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/- 0.5 microM 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 preQ(1)-tRNA(Tyr) when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ(1)-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 preQ(1)-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.
Department of Chemistry, Portland State University, Portland, Oregon 97207-0751, USA.
Steven G
SG
Van Lanen
Dirk
D
Iwata-Reuyl
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007535:Isomerases; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009704:Nucleoside Q / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000494:pharmacology
D009704:Nucleoside Q; D012266:Ribose; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; D012435:S-Adenosylhomocysteine; D010430:Pentosyltransferases; C082572:S-adenosylmethionine - tRNA ribosyltransferase-isomerase; D007535:Isomerases; D000241:Adenosine; C006235:sinefungin