Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction.
Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Garcia
G A
GA
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
Leatherbarrow
R J
RJ
Eckstein
F
F
Fersht
A R
AR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D013873:Thionucleotides / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
D008670:Metals; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D002104:Cadmium; D003035:Cobalt; C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D008274:Magnesium